Methods using hdac11 inhibitors

ABSTRACT

The present invention provides methods and uses of inhibitors of histone deacetylase 11 (HDAC11) in the treatment of diseases and/or disorders, such as, for example, cell proliferative diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/410,766 filed Oct. 20, 2016, 62/410,767 filed Oct. 20, 2016, and62/410,768 filed Oct. 20, 2016, the contents of all of which are herebyincorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created, Oct. 19, 2017, is namedFOTH039_ST25.txt and is 10.6 KB in size.

FIELD OF THE INVENTION

The present invention relates to inhibitors of histone deacetylase 11(HDAC11) useful in the treatment of certain diseases and/or disorders,including diseases and disorders associated with cell proliferation(e.g., cancers) and/or diseases and disorders associated with increasedexpression of genes associated with stem cell activity. The presentdisclosure provides, at least in part, methods and compositions fortreating or preventing diseases and/or disorders that include a HDAC11inhibitor.

BACKGROUND OF THE INVENTION

Diseases associated with cell proliferation, such as cancer, remain aserious public health problem. For example, about 595,690 people in theUnited States of America are expected to die of cancer in 2016 aloneaccording to the American Cancer Society, Cancer Facts & Figures 2016(www.cancer.org/acs/groups/content/@research/documents/document/acspc-047079.pdf).While many targets have been identified as associated with certaincancers, it has been challenging to develop selective therapies.Accordingly, there remains a need for effective, safe and selectivemethods of treating diseases and/or disorders associated with cellproliferation.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides compounds and methods for treatment orprevention of diseases and/or disorders associated with cellproliferation and/or associated with amplification of genes associatedwith stem cell activity. The present disclosure encompasses therecognition that HDAC11 inhibitors may have potential benefit fortreatment of diseases and/or disorders associated with cellproliferation and/or associated with activation (e.g., amplification) ofgenes associated with stem cell activity. In some embodiments, a diseaseor disorder associated with cell proliferation and/or associated withactivation (e.g., amplification) of genes associated with stem cellactivity is a cancer.

Cancers for treatment with a HDAC11 inhibitor include, but are notlimited to, gastro-intestinal cancers, nervous system cancers,esophageal squamous cell carcinomas, oral squamous cell carcinomas, skincancers, lung cancers, breast cancers, squamous cell carcinomas, lungadenocarcinomas, non-small cell lung cancer, small cell lung cancer,prostate cancers, sinonasal cancers, leukemias, lymphomas (Hodgkin's andnon-Hodgkin's), myelomas and myeloproliferative disorders. In someembodiments, a cancer is associated with activation of JAK/STATsignaling. In some embodiments, a cancer is associated with activationof a Signal Transducer and Activator of Transcription (STAT), such as,for example, STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B),and/or STAT6. In some embodiments, activation of a STAT transcriptionfactor is associated with an increase in gene expression and/or adecrease in protein phosphorylation. In some embodiments, a cancer isassociated with activation of STAT3. In some certain embodiments, acancer associated with activation of STAT3 is breast cancer. In someembodiments, a cancer is associated with activation of SRY (sexdetermining region Y)-box 2 (SOX2). In some embodiments, activation ofSOX2 is an increase in SOX2 gene expression.

The present disclosure provides methods of treating cancer, wherein oneor more cancer cells exhibit stem cell-like properties. In someembodiments, methods comprise administering to a patient in need thereofan effective amount of a HDAC11 inhibitor. In some embodiments, one ormore cancer cells that exhibit stem cell-like properties is associatedwith increased expression (e.g., gene amplification) or activity of amarker for a cancer stem cell. In some embodiments, a marker for acancer stem cell is activation of one or more components of a JAK/STATsignaling pathway. In some embodiments, a marker for a cancer stem cellis increased expression (e.g., gene amplification) or activity of SOX2.In some embodiments, a marker for a cancer stem cell is increasedexpression (e.g., gene amplification) or activity of STAT3.

In some embodiments, the present disclosure provides methods of treatingdiseases and/or disorders associated with HDAC11. Diseases and/ordisorders associated with HDAC11 include, e.g., cell proliferationdiseases or disorders having cancer cells that exhibit cancer stem cellproperties and/or are associated with amplification of genes associatedwith cancer stem cell activity.

In some embodiments, a disease and/or disorder for treatment with aHDAC11 inhibitor is a myeloproliferative neoplasm (MPN). In someembodiments, MPNs for treatment with a HDAC11 inhibitor include, but arenot limited to, polycythemia vera (PV), essential thrombocythemia (ET)and primary myelofibrosis (PMF).

In some embodiments, a cell responsive to treatment with a HDAC11inhibitor is a cancer stem cell (CSC). In some embodiments, a cellresponsive to treatment with a HDAC11 inhibitor is cell associated withexpression of a CSC marker. In some embodiments, a CSC marker isactivation of one or more components of a JAK/STAT signaling pathway(e.g., SOX2 and/or STAT3).

In some embodiments, the present disclosure provides a method ofinhibiting and/or reducing proliferation of a stem cell-like cancer cellin a patient having cancer, comprising administering to a HDAC11inhibitor as described herein to the patient. In some embodiments, astem cell-like cancer is associated with activation of JAK/STATsignaling. In some embodiments, a stem cell-like cancer is associatedwith a gene amplification of SOX2. In some embodiments, a stem cell-likecancer is associated with activation of STAT3.

In some embodiments, a cancer is a lung cancer (e.g., adenocarcinoma ofthe lung, NSCLC), a hematological cancer (e.g., a leukemia, a lymphoma,a myeloma and a myeloproliferative disorder), a skin cancer, a breastcancer, a squamous cell carcinoma, a gastro-intestinal cancer (e.g.,esophageal cancer, gastric cancer), a uterine cancer, a prostrate cancerand/or a nervous system cancer.

In some embodiments, the present disclosure provides a method oftreating a hematological cancer comprising administering to a patient aneffective amount of a HDAC11 inhibitor. In some embodiments, ahematological cancer is a myeloproliferative disorder, a lymphoma, aleukemia, and/or myeloma. In some embodiments, a hematological cancer isa myeloproliferative disorder. In certain embodiments, amyeloproliferative disorder is polycythemia vera, essentialthrombocytemia, myelofibrosis, chronic myelogenous leukemia, chronicneutrophilic leukemia, chronic eosinophilic leukemia, or mastocytosis.

In some embodiments, a HDAC11 inhibitor is a siRNA, a shRNA, an antibodyagent, or a chemical compound (e.g., a small molecule). In someembodiments, a HDAC11 inhibitor is a chemical compound that is a smallmolecule. In some embodiments, a small molecule HDAC11 inhibitor is atleast 10-fold selective for the inhibition of HDAC11 over one or moreother histone deacetylase isoforms (e.g., HDAC1, HDAC2, HDAC3, HDAC4,HDAC5, HDAC6, HDAC7, HDAC8, HDAC9 and/or HDAC10). In some embodiments, asmall molecule HDAC11 inhibitor is at least 10-fold, 20-fold, 30-fold,40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold, 2,000-fold,3,000-fold, or more selective for inhibition of HDAC11 over one or moreother histone deacetylase isoforms (e.g., HDAC1, HDAC2, HDAC3, HDAC4,HDAC5, HDAC6, HDAC7, HDAC8, HDAC9 and/or HDAC10). In some embodiments, asmall molecule HDAC11 inhibitor is at least 10-fold selective for theinhibition of HDAC11 over each of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,HDAC6, HDAC7, HDAC8, HDAC9 and HDAC10. In some embodiments, a HDAC11inhibitor is specific for huma HDAC11.

The present disclosure also encompasses the recognition that inhibitionof HDAC11 may be beneficial for treatment or prevention of inflammatoryand/or autoimmune diseases. In some embodiments, a disease or disorderfor treatment with a HDAC11 inhibitor is an immune or inflammatorydisorder, which may be acute or chronic. In some embodiments, a diseaseor disorder for treatment with a HDAC11 inhibitor is inflammatory boweldisease, psoriasis, rheumatoid arthritis, multiple sclerosis, and/orAlzheimer's disease.

In some embodiments, methods described herein may include administrationof one, two, three or more HDAC11 inhibitors.

In some embodiments, methods described herein may further includeadministering a JAK2 inhibitor to a patient in need thereof. In someembodiments, a JAK2 inhibitor is a siRNA, a shRNA, an antibody agent, ora chemical compound. In certain embodiments, a JAK2 inhibitor is JAK2inhibitor is ruxolitinib, baricitinib, CYT387, lestaurtinib, orpacritinib.

In some embodiments, a HDAC11 inhibitor is administered to a patientthat has been or will be administered a JAK2 inhibitor, such that thepatient receives treatment with both. In some embodiments, a JAK2inhibitor is administered to a patient that has been or will beadministered a HDAC11 inhibitor, such that the patient receivestreatment with both.

In some embodiments, the present disclosure provides a method oftreating a patient having a myeloproliferative disorder that includeadministering to a patient in need thereof a HDAC11 inhibitor and a JAK2inhibitor. In some embodiments, a myeloproliferative disorder ispolycythemia vera, essential thrombocytemia, myelofibrosis, chronicmyelogenous leukemia, chronic neutrophilic leukemia, chroniceosinophilic leukemia, or mastocytosis.

In some embodiments, methods described herein may further compriseadministering a hedgehog pathway inhibitor to the patient. In someembodiments, a hedgehog pathway inhibitor is vismodegib, erismodegib,BMS-833923, glasdegib, taladegib, or saridegib. In some embodiments, aHDAC11 inhibitor is administered to a patient that has been or will beadministered a hedgehog pathway inhibitor, such that the patientreceives treatment with both. In some embodiments, a hedgehog pathwayinhibitor is administered to a patient that has been or will beadministered a HDAC11 inhibitor, such that the patient receivestreatment with both.

In certain embodiments, a HDAC11 inhibitor is a siRNA, a shRNA, anantibody agent, or a chemical compound. In some embodiments, a chemicalcompound is a small molecule that is at least 10-fold selective for theinhibition of HDAC11 over one or more other histone deacetylase isoforms(e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9and/or HDAC10).

In some embodiments, the present disclosure provides a method fortreating a patient having a myeloproliferative disorder resistant to aJAK2 inhibitor, where the method includes administering a HDAC11inhibitor to the patient. In some embodiments, a myeloproliferativedisorder is polycythemia vera, essential thrombocytemia, myelofibrosis,chronic myelogenous leukemia, chronic neutrophilic leukemia, chroniceosinophilic leukemia, or mastocytosis.

In certain embodiments, the HDAC11 inhibitor is a siRNA, a shRNA, anantibody agent, or a chemical compound. In some embodiments, a chemicalcompound is a small molecule that is at least 10-fold selective for theinhibition of HDAC11 over one or more other histone deacetylase isoforms(e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9and/or HDAC10).

In some embodiments, the present disclosure provides a method oftreating a patient having a myeloproliferative disorder, where themethod includes administering to a patient a HDAC11 inhibitor and ahedgehog pathway inhibitor. In some embodiments, a myeloproliferativedisorder is polycythemia vera, essential thrombocytemia, myelofibrosis,chronic myelogenous leukemia, chronic neutrophilic leukemia, chroniceosinophilic leukemia, or mastocytosis. In certain embodiments, a HDAC11inhibitor is a siRNA, a shRNA, an antibody agent, or a chemicalcompound. In some embodiments, a chemical compound is a small moleculethat is at least 10-fold selective for the inhibition of HDAC11 overother histone deacetylase isoforms (e.g., HDAC1, HDAC2, HDAC3, HDAC4,HDAC5, HDAC6, HDAC7, HDAC8, HDAC9 and/or HDAC10). In some embodiments, ahedgehog pathway inhibitor is vismodegib, erismodegib, BMS-833923,glasdegib, taladegib, or saridegib.

In some embodiments, the present disclosure provides a method oftreating cancer wherein one or more cancer cells exhibit stem cell-likeproperties, where the method includes treating a patient with a firstline therapy and administering to the patient a HDAC11 inhibitor,whereby any cancer cells surviving from the first line therapy arereduced or eliminated after treatment with the HDAC11 inhibitor.

In some embodiments, a cancer is a lung cancer (e.g., adenocarcinoma ofthe lung, NSCLC), a hematological cancer (e.g., a leukemia, a lymphoma,a myeloma and a myeloproliferative disorder), a skin cancer, a breastcancer, a squamous cell carcinoma, gastro-intestinal cancer (e.g.,esophageal cancer, gastric cancer), a uterine cancer, a prostrate cancerand/or a nervous system cancer. In some embodiments, a cancer isesophageal squamous cell carcinoma, oral squamous cell carcinoma, lungsquamous cell carcinoma, lung adenocarcinoma, non-small cell lungcancer, small cell lung cancer and/or sinonasal cancer. In certainembodiments, the cancer is breast cancer. In certain embodiments, aHDAC11 inhibitor is a siRNA, a shRNA, an antibody agent, or a chemicalcompound. In some embodiments, a chemical compound is a small moleculethat is at least 10-fold selective for the inhibition of HDAC11 overother histone deacetylase isoforms (e.g., HDAC1, HDAC2, HDAC3, HDAC4,HDAC5, HDAC6, HDAC7, HDAC8, HDAC9 and/or HDAC10). In certainembodiments, a first line therapy is resection, radiation, and/or stemcell transplant.

In some embodiments, a HDAC11 compound used in accordance with thepresent disclosure is of Formulae I, I′, or II, as defined herein.

In some embodiments, the present disclosure provides a compound ofFormula I:

wherein each of Y₁, Y₂, Y₃, Q, L, Z, X₁, X₂, X₃, and X₄ is as definedherein.

In some embodiments, the present disclosure provides a compound ofFormula I′:

wherein each of Y₁, Y₂, Y₃, Y₄, Q, L, Z, X₁, X₂, X₃, and X₄ is asdefined herein.

In some embodiments, the present disclosure provides a compound ofFormula II:

wherein each of Y¹, Y², Y³, R, L, m, n, q, r, X¹, X², X³, X⁴, X⁵, and X⁶is as defined herein.

BRIEF DESCRIPTION OF THE DRAWING

The Drawing included herein, which is composed of the following Figures,is for illustration purposes only and not for limitation.

FIG. 1 shows that disease burden was reduced by HDAC11 deficiency in abone marrow transplant MPN model. (A) depicts a schematic representationof the experimental protocol and (B) depicts platelet and red blood cellcounts and images of spleens from mice.

FIG. 2 shows that STAT3 and STAT5 phosphorylation were decreased byHDAC11 deficiency in a bone marrow transplant MPN model.

FIG. 3 shows that growth of MPL- or JAK2-mutant expressing cell lineswas inhibited by HDAC11 inhibitors.

FIG. 4 shows that growth of HEL92.1.7 and SET-2 cell lines was inhibitedby HDAC11 inhibitors and ruxolitinib.

FIG. 5 shows G1 cell cyle arrest in HEL92.1.7 cells after treatment withan HDAC11 inhibitor.

FIG. 6 shows inhibition of STAT3 and STAT5 phosphorylation in HEL92.1.7cells after treatment with a HDAC11 inhibitor.

FIG. 7 shows inhibition of colony growth from (A) erythroid and (B)myeloid patient-derived cells after treatment with a HDAC11 inhibitor.

FIG. 8 shows growth inhibition of (A) A549 and (B) H1650 stem-likecancer cells with increasing concentrations of HDAC11 inhibitors. Foreach sample: control—leftmost column, 0.5 μM—second column from left,1.25 μM—third column from left (center), 2.5 μM-second column fromright, 5.0 μM—rightmost column.

FIG. 9 shows inhibition of tubule formation in H1650 SP cells by HDAC11inhibitors.

FIG. 10 shows that HDAC11 inhibitors impaired A549 cell migration.

FIG. 11 shows that HDAC11 and HDAC1 knockdown resulted in decreasedexpression of SOX2. For each gene (Sox2, Oct4 and Nanog) fold change inmRNA levels was measured after knockdown with siRNAs: control—leftmostcolumn, HDAC1 siRNA—second column from left, HDAC6 siRNA—second columnfrom right, HDAC11 siRNA—rightmost column.

FIG. 12 shows that treatment with HDAC11 inhibitors resulted indecreased expression of SOX2 in (A) A549 and (B) H1650 cell lines. Forcontrol and each HDAC11 inhibitor, fold change in mRNA levels for eachgene was measures: Gli1—leftmost column, Gli2—second column from left,Sox2—second column from right, YAP1—rightmost column.

FIG. 13 shows that HDAC11 inhibitor compounds inhibited growth of H1650cell lines co-cultured with cancer associated fibroblasts.

FIG. 14 shows that a combination of HDAC11 inhibitors and Smoothenedinhibitors inhibited growth of (A) A549 and (B) H2170 cells.

FIG. 15 shows several exemplary HDAC11 inhibitors.

DETAILED DESCRIPTION

HDAC11 interacts with or regulates RNA splicing-related proteins (e.g.,SMN1, Dicer1, Gemin3, and Gemin4), cell cycle-related proteins (e.g.,Cdt1, BubR1, and Cdc25), and immune cell signaling proteins (e.g., IL-10and OX40 ligand). Accordingly, inhibition of HDAC11 can have downstreameffects that can play a role in the development of certain diseases suchas cell proliferative diseases. The present disclosure provides methodsfor using inhibitors of HDAC11 to treat cell proliferative diseases anddisorders including MPNs, hematological malignancies, and solid tumormalignancies. The present disclosure also provides methods for usinginhibitors of HDAC11 to treat cell proliferation diseases or disordershaving cancer cells that exhibit cancer stem cell properties and/or areassociated with amplification of genes associated with cancer stem cellactivity (e.g., SOX2 and STAT3).

The present invention is based in part on the discovery that HDAC11inhibitors prevent growth and survival of JAK2 and myeloproliferativeleukemia gene (MPL) mutant cell lines and patient-derived samples.Furthermore, as discussed in detail herein, knockout of HDAC11 reducesthrombocytosis and erythrocytosis in a MPN model using MPL mutant bonemarrow transplant mice. The present invention is also based in part onthe discovery that HDAC11 inhibitors prevent the growth of stemcell-like cancer cells and also decrease the expression of certain stemcell transcription factors such as SOX2 and STAT3.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. All patents and publications cited in thisspecification are incorporated herein by reference in their entireties.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

Certain Definitions

The articles “a” and “an” are used in this disclosure to refer to one ormore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “and/or” is used in this disclosure to mean either “and” or“or” unless indicated otherwise.

The term “inhibitor” as used herein encompasses molecules that block,inhibit, and/or decrease the expression or activity of HDAC11. As usedherein, an inhibitor may be a polypeptide, polynucleotide, antibody, ora chemical compound. Examples of HDAC11 activity include deacetylasefunction and associating with a protein complex or transcriptionregulator, such as a promoter.

The phrases “interacts with” and “associates with” are usedinterchangeably herein to describe proteins that form a multi-proteincomplex via non-covalent interactions. In some embodiments, HDAC11co-precipitates with a protein with which it is associated.

An “effective amount” when used in connection with an inhibitor is anamount effective for treating or preventing a disease in a subject asdescribed herein.

The term “carrier”, as used in this disclosure, encompasses carriers,excipients, and diluents and means a material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting apharmaceutical agent (e.g., HDAC11 inhibitor or JAK2 inhibitor) from oneorgan, or portion of the body, to another organ, or portion of the bodyof a subject.

The term “treating” with regard to a subject, refers to improving atleast one symptom of the subject's disorder. Treating includes curing,improving, or at least partially ameliorating the disorder and/orsymptoms related to the disorder.

The term “disorder” is used in this disclosure to mean, and is usedinterchangeably with, the terms disease, condition, or illness, unlessotherwise indicated.

The terms “cancer”, “malignancy”, “neoplasm”, “tumor”, and “carcinoma”,are used herein to refer to cells that exhibit relatively abnormal,uncontrolled, and/or autonomous growth, so that they exhibit an aberrantgrowth phenotype characterized by a significant loss of control of cellproliferation. In some embodiments, a tumor may be or comprise cellsthat are precancerous (e.g., benign), malignant, pre-metastatic,metastatic, and/or non-metastatic. The present disclosure specificallyidentifies certain cancers to which its teachings may be particularlyrelevant. In some embodiments, a relevant cancer may be characterized bya solid tumor. In some embodiments, a relevant cancer may becharacterized by a hematologic tumor. In general, examples of differenttypes of cancers known in the art include, for example, hematopoieticcancers including leukemias, lymphomas (Hodgkin's and non-Hodgkin's),myelomas and myeloproliferative disorders; sarcomas, melanomas,adenomas, carcinomas of solid tissue, squamous cell carcinomas of themouth, throat, larynx, and lung, liver cancer, genitourinary cancerssuch as prostate, cervical, bladder, uterine, and endometrial cancer andrenal cell carcinomas, bone cancer, pancreatic cancer, skin cancer,cutaneous or intraocular melanoma, cancer of the endocrine system,cancer of the thyroid gland, cancer of the parathyroid gland, head andneck cancers, breast cancer, gastro-intestinal cancers and nervoussystem cancers, benign lesions such as papillomas, and the like.

The term “administer”, “administering”, or “administration” as used inthis disclosure refers to either directly administering a therapeuticagent such as a compound or pharmaceutically acceptable salt of thecompound or a composition to a subject, or administering a prodrugderivative or analog of the therapeutic agent to the subject, which canform an equivalent amount of active compound within the subject's body.

A “patient” or “subject” is a mammal, e.g., a human, mouse, rat, guineapig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey,chimpanzee, baboon or rhesus.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. While perfect complementarity is oftendesired, some embodiments can include one or more (e.g., 10, 9, 8, 7, 6,5, 4, 3, 2, or 1) mismatches with respect to the target RNA. Variationsat any location within the oligomer are included. In certainembodiments, variations in sequence near the termini of an oligomer aregenerally preferable to variations in the interior, and if present, aremay be within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides of the5′ and/or 3′ terminus.

By “isolated” is meant a substance and/or entity that has been (1)separated from at least some of the components with which it wasassociated when initially produced (whether in nature and/or in anexperimental setting), and/or (2) designed, produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% of the other components with which they wereinitially associated. In some embodiments, an isolated substance orentity is “pure”. As used herein, a substance or entity is “pure” if itis substantially or essentially free from components that normallyaccompany it in its native state. In some embodiments, a substanceand/or entity is about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or more than about 99% pure. For example, an “isolatedpolynucleotide” or “isolated oligonucleotide,” as used herein, may referto a polynucleotide that has been purified or removed from the sequencesthat flank it in a naturally-occurring state, e.g., a DNA fragment thathas been removed from the sequences that are normally adjacent to thefragment. In another example, an “isolated polypeptide” or “isolatedantibody,” as used herein, may refer to a polypeptide (e.g., anantibody) that has been purified or removed from one or more componentsof its naturally-occurring environment.

The term “reduce” or “inhibit” may relate generally to the ability ofone or more inhibitors of the invention to “decrease” a relevantbiological activity or cellular response, such as catalytic activity ora symptom of a disease or condition described herein, as measuredaccording to routine techniques in the diagnostic art. Relevantphysiological or cellular responses (in vivo or in vitro) will beapparent to persons skilled in the art, and may include reductions inthe symptoms or pathology of MPNs. A “decrease” in a biological activitymay be “statistically significant” as compared to the biologicalactivity produced by no inhibitor or a control composition, and mayinclude a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including allintegers in between.

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395). In this way sequences ofa similar or substantially different length to those cited herein couldbe compared by insertion of gaps into the alignment, such gaps beingdetermined, for example, by the comparison algorithm used by GAP.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” and “substantial identity”. A “reference sequence” is atleast 8 or 10 but frequently 15 to 18 and often at least 25 monomerunits, inclusive of nucleotides and amino acid residues, in length.Because two polynucleotides may each comprise (1) a sequence (i.e., onlya portion of the complete polynucleotide sequence) that is similarbetween the two polynucleotides, and (2) a sequence that is divergentbetween the two polynucleotides, sequence comparisons between two (ormore) polynucleotides are typically performed by comparing sequences ofthe two polynucleotides over a “comparison window” to identify andcompare local regions of sequence similarity. A “comparison window”refers to a conceptual segment of at least 6 contiguous positions,usually about 50 to about 100, more usually about 100 to about 150 inwhich a sequence is compared to a reference sequence of the same numberof contiguous positions after the two sequences are optimally aligned.The comparison window may comprise additions or deletions (i.e., gaps)of about 20% or less as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerized implementations of algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Drive Madison,Wis., USA) or by inspection and the best alignment (i.e., resulting inthe highest percentage homology over the comparison window) generated byany of the various methods selected. Reference also may be made to theBLAST family of programs as for example disclosed by Altschul et al.,1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequenceanalysis can be found in Unit 19.3 of Ausubel et al., “Current Protocolsin Molecular Biology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.

“dsRNA” refers to a ribonucleic acid molecule having a duplex structurecomprising two complementary and anti-parallel nucleic acid strands(i.e., the sense and antisense strands). Not all nucleotides of a dsRNAmust exhibit Watson-Crick base pairs; the two RNA strands may besubstantially complementary. The RNA strands may have the same or adifferent number of nucleotides. The term “dsRNA” also includes “siRNA”or short interfering RNA.

By “vector” or “nucleic acid construct” is meant a polynucleotidemolecule, preferably a DNA molecule derived, for example, from aplasmid, bacteriophage, yeast or virus, into which a polynucleotide canbe inserted or cloned. A vector preferably contains one or more uniquerestriction sites and can be capable of autonomous replication in adefined host cell including a target cell or tissue or a progenitor cellor tissue thereof, or be integrable with the genome of the defined hostsuch that the cloned sequence is reproducible. Accordingly, the vectorcan be an autonomously replicating vector, i.e., a vector that exists asan extra-chromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. The vector can contain any means for assuringself-replication. Alternatively, the vector can be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated.

A wild-type gene or gene product is that which is most frequentlyobserved in a population and is thus arbitrarily designed the “normal”or “wild-type” form of the gene.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B. and March,J., John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

The term “alkylaryl” refers to aryl groups connected to an adjacentalkyl wherein the linkage is located at the alkyl end. A“(C₁-C₆)alkylaryl” refers to a group that contains a C₁-C₆ alkyl groupbonded to an aryl group. Accordingly, groups such as benzyl,phenylethyl, or mesitylenyl constitute exemplary representatives ofalkylaryl of the present invention.

The term “aryl” refers to cyclic, aromatic hydrocarbon groups that have1 to 2 aromatic rings, including monocyclic or bicyclic groups such asphenyl, biphenyl or naphthyl. Where containing two aromatic rings(bicyclic, etc.), the aromatic rings of the aryl group may be joined ata single point (e.g., biphenyl), or fused (e.g., naphthyl). The arylgroup may be optionally substituted by one or more substituents, e.g., 1to 5 substituents, at any point of attachment. Exemplary substituentsinclude, but are not limited to, —H, -halogen, —O—C₁-C₆alkyl,—C₁-C₆alkyl, —OC₂-C₆alkenyl, —OC₂-C₆alkynyl, —C₂-C₆alkenyl,—C₂-C₆alkynyl, —OH, —OP(O)(OH)₂, —OC(O)C₁-C₆alkyl, —C(O)C₁-C₆alkyl,—OC(O)OC₁-C₆alkyl, —NH₂, —NH(C₁-C₆alkyl), —N(C₁-C₆alkyl)₂,—S(O)₂—C₁-C₆alkyl, —S(O)NHC₁-C₆alkyl, and —S(O)N(C₁-C₆alkyl)₂. Thesubstituents can themselves be optionally substituted. Furthermore whencontaining two fused rings the aryl groups herein defined may have asaturated, unsaturated or partially saturated ring fused with anaromatic ring. Exemplary ring systems of these aryl groups includeindanyl, indenyl, tetrahydronaphthalenyl, and tetrahydrobenzoannulenyl.

Unless otherwise specifically defined, “heteroaryl” means a monovalentmonocyclic aromatic radical or a polycyclic aromatic radical of 5 to 24ring atoms, containing one or more ring heteroatoms selected from N, S,P, and O, the remaining ring atoms being C. Heteroaryl as herein definedalso means a bicyclic heteroaromatic group wherein the heteroatom isselected from N, S, P, and O. The aromatic radical is optionallysubstituted independently with one or more substituents describedherein. Examples include, but are not limited to, furyl, thienyl,pyrrolyl, pyridyl, pyrazolyl, pyrimidinyl, imidazolyl, isoxazolyl,oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl,benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole,benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl,imidazo[1,2-b]pyrazolyl, furo[2,3-c]pyridinyl, imidazo[1,2-a]pyridinyl,indazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2-c]pyridinyl,pyrazolo[3,4-c]pyridinyl, thieno[3,2-c]pyridinyl,thieno[2,3-c]pyridinyl, thieno[2,3-b]pyridinyl, benzothiazolyl, indolyl,indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuranyl,benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl,dihydrobenzothiazine, dihydrobenzoxanyl, quinolinyl, isoquinolinyl,1,6-naphthyridinyl, benzo[de]isoquinolinyl,pyrido[4,3-b][1,6]naphthyridinyl, thieno[2,3-b]pyrazinyl, quinazolinyl,tetrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, isoindolyl,pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl,pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b]pyridinyl,pyrrolo[1,2-a]pyrimidinyl, tetrahydro pyrrolo[1,2-a]pyrimidinyl,3,4-dihydro-2H-1λ²-pyrrolo[2,1-b]pyrimidine, dibenzo[b,d] thiophene,pyridin-2-one, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl,1H-pyrido[3,4-b][1,4] thiazinyl, benzooxazolyl, benzoisoxazolyl,furo[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl,furo[3,2-b]pyridine, [1,2,4]triazolo[1,5-a]pyridinyl, benzo[1,2,3]triazolyl, imidazo[1,2-a]pyrimidinyl,[1,2,4]triazolo[4,3-b]pyridazinyl, benzo[c][1,2,5]thiadiazolyl,benzo[c][1,2,5]oxadiazole, 1,3-dihydro-2H-benzo[d]imidazol-2-one,3,4-dihydro-2H-pyrazolo [1,5-b][1,2]oxazinyl,4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl, thiazolo[5,4-d]thiazolyl,imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2,3-b]pyrrolyl, 3H-indolyl,and derivatives thereof. Furthermore when containing two fused rings theheteroaryl groups herein defined may have a saturated, unsaturated orpartially saturated ring fused with an aromatic ring. Exemplary ringsystems of these heteroaryl groups include indolinyl, indolinonyl,dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl,tetrahydroquinolinyl, dihydrobenzothiazine,3,4-dihydro-1H-isoquinolinyl, 2,3-dihydrobenzofuran, indolinyl, indolyl,and dihydrobenzoxanyl.

“Alkyl” refers to a straight or branched chain saturated hydrocarboncontaining 1-12 carbon atoms. C₁-C₆alkyl groups contain 1 to 6 carbonatoms. Examples of a C₁-C₆alkyl group include, but are not limited to,methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec-butyl andtert-butyl, isopentyl and neopentyl.

“Alkenyl” refers to a straight or branched chain unsaturated hydrocarboncontaining 2-12 carbon atoms. The “alkenyl” group contains at least onedouble bond in the chain. The double bond of an alkenyl group can beunconjugated or conjugated to another unsaturated group. Examples ofalkenyl groups include ethenyl, propenyl, n-butenyl, iso-butenyl,pentenyl, or hexenyl. An alkenyl group can be unsubstituted orsubstituted. Alkenyl, as herein defined, may be straight or branched.

“Alkynyl” refers to a straight or branched chain unsaturated hydrocarboncontaining 2-12 carbon atoms. The “alkynyl” group contains at least onetriple bond in the chain. Examples of alkenyl groups include ethynyl,propanyl, n-butynyl, iso-butynyl, pentynyl, or hexynyl. An alkynyl groupcan be unsubstituted or substituted.

The term “cycloalkyl” means monocyclic or polycyclic saturated carbonrings containing 3-18 carbon atoms. Examples of cycloalkyl groupsinclude, without limitations, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl,bicyclo[2.2.2]octanyl, or bicyclo[2.2.2]octenyl. A C₃-C₈ cycloalkyl is acycloalkyl group containing between 3 and 8 carbon atoms. A cycloalkylgroup can be fused (e.g., decalin) or bridged (e.g., norbornane).

The term “cycloalkenyl” means monocyclic, non-aromatic unsaturatedcarbon rings containing 4-18 carbon atoms. Examples of cycloalkenylgroups include, without limitation, cyclopentenyl, cyclohexenyl,cycloheptenyl, cyclooctenyl, and norborenyl. A C₄-C₈ cycloalkenyl is acycloalkenyl group containing between 4 and 8 carbon atoms.

The terms “heterocyclyl” or “heterocycloalkyl” or “heterocycle” refer tomonocyclic or polycyclic 3 to 24-membered non-aromatic rings containingcarbon and heteroatoms taken from oxygen, phosphorous, nitrogen, orsulfur and wherein there is not delocalized π electrons (aromaticity)shared among the ring carbon or heteroatoms. Heterocyclyl rings include,but are not limited to, oxetanyl, azetadinyl, tetrahydrofuranyl,pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl,pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl,morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinylS-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, andhomotropanyl. A heteroycyclyl or heterocycloalkyl ring can also be fusedor bridged, e.g., can be a bicyclic ring.

As used herein, the term “halo” or “halogen” means a fluoro, chloro,bromo, or iodo group.

The term “carbonyl” refers to a functional group composing a carbon atomdouble-bonded to an oxygen atom. It can be abbreviated herein as “oxo”,as C(O), or as C═O.

“Spirocycle” or “spirocyclic” means carbogenic bicyclic ring systemswith both rings connected through a single atom. The rings can bedifferent in size and nature, or identical in size and nature. Examplesinclude spiropentane, spirohexane, spiroheptane, spirooctane,spirononane, or spirodecane. One or both of the rings in a spirocyclecan be fused to another carbocyclic, heterocyclic, aromatic, orheteroaromatic ring. A C₅-C₁₂ spirocycle is a spirocycle containingbetween 5 and 12 carbon atoms.

The term “spirocycloalkenyl” means a carbogenic bicyclic ring systemcontaining 5-12 atoms with both ring systems connected through a singleatom and wherein at least one ring contains a carbon-carbon double bond.The rings can be different in size and nature, or identical in size andnature. One or both rings may contain a double-bond. One or both of therings in a spirocycloalkenyl can further be fused to anothercarbocyclic, heterocyclic, aromatic, or heteroaromatic ring.

The term “spirocyclic heterocycle,” “spiroheterocyclyl,” or“spiroheterocycle” is understood to mean a spirocycle wherein at leastone of the rings is a heterocycle (e.g., at least one of the rings isfuranyl, morpholinyl, or piperadinyl). A spirocyclic heterocycle cancontain between 5 and 12 atoms, at least one of which is a heteroatomselected from N, O, S and P.

The disclosure also includes pharmaceutical compositions comprising aneffective amount of a disclosed compound and a pharmaceuticallyacceptable carrier. Representative “pharmaceutically acceptable salts”include, e.g., water-soluble and water-insoluble salts, such as theacetate, amsonate (4,4-diaminostilbene-2,2-disulfonate),benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate,bromide, butyrate, calcium, calcium edetate, camsylate, carbonate,chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate,estolate, esylate, fiunarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, sethionate,lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate,mesylate, methylbromide, methylnitrate, methylsulfate, mucate,napsylate, nitrate, N-methylglucamine ammonium salt,3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate(1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate,phosphate/diphosphate, picrate, polygalacturonate, propionate,p-toluenesulfonate, salicylate, stearate, subacetate, succinate,sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate,tosylate, triethiodide, and valerate salts.

Histone Deacetylases and HDAC11

Histone deacetylases (HDAC, EC number 3.5.1) are a group of hydrolasesremoving the acetyl group from an ε-N-acetyl lysine amino acid of ahistone or other substrate protein. Depending on sequence identity anddomain organization, HDACs have been classified into class I (includingHDAC1-3 and 8), class Ila (HDAC4, 5, 7, 9), class lib (HDAC6 and 10),class III (including sirtuins) and class IV (HDAC11) (Dokmanovic et al,2007, Mol Cancer Res October 5; 981-989).

HDAC11 is a class IV histone deacetylase (HDAC). HDAC11 is highlyexpressed in brain, heart, skeletal muscle, kidney and testis andprimarily localized in the nucleus (Gao et al, J Biol Chem. 2002, Jul.12; 277(28):25748-55). Without wishing to be bound by theory, histonedeacetylation may provide a tag for epigenetic repression and play animportant role in transcriptional regulation, cell cycle progression anddevelopmental events. Additionally, HDAC substrates are not restrictedto histones, and HDACs have been shown to function in numerous cellregulatory pathways. For example, HDAC11 has been reported todeacetylate or associate with cell cycle-related proteins includingCdt1, Geminin (Wong et al, Cell Cycle. 2010, November 1; 9(21):4351-63),BubR1 (Watanabe et al, Cell Rep. 2014, Apr. 24; 7(2):552-64), and Cdc25(Lozada et al, Oncotarget. 2016, Mar. 7). HDAC11 was also reported tofunction in RNA splicing as part of the survival of motor neuron (SMN)complex in association with SMN1, Dicer1, Gemin3, and Gemin4 (Joshi etal, Mol Syst Biol. 2013, 9:672). HDAC11 has also been reported tofunction in regulating dendrite growth (Watanabe et al, Cell Rep. 2014,Apr. 24; 7(2):552-64).

HDAC11 may play a role in certain cancers (Deubzer et al, Int J Cancer.2013, May 1; 132(9):2200-8; WO 2005/071079; WO 2007/038073), such as,for example, Hodgkin lymphoma (Buglio et al, Blood. 2011, Mar. 10;117(10):2910-7).

Alternative splicing of HDAC11 results in multiple transcript variants.In some embodiments, an inhibitor of HDAC11 may selectively inhibit oneor more variants of HDAC11.

In some embodiments, an inhibitor of HDAC11 selectively inhibitsactivity of a HDAC11 that is or comprises a sequence of any one of SEQID NO: 13-15. In some embodiments, an inhibitor of HDAC11 selectivelyinhibits activity of a HDAC11 that is or comprises a sequence that is80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NO: 13-15.

human HDAC11 isoform 1 SEQ ID NO: 13MLHTTQLYQH VPETRWPIVY SPRYNITFMG LEKLHPFDAGKWGKVINFLK EEKLLSDSML VEAREASEED LLVVHTRRYLNELKWSFAVA TITEIPPVIF LPNFLVQRKV LRPLRTQTGGTIMAGKLAVE RGWAINVGGG FHHCSSDRGG GFCAYADITLAIKFLFERVE GISRATIIDL DAHQGNGHER DFMDDKRVYIMDVYNRHIYP GDRFAKQAIR RKVELEWGTE DDEYLDKVERNIKKSLQEHL PDVVVYNAGT DILEGDRLGG LSISPAGIVKRDELVFRMVR GRRVPILMVT SGGYQKRTAR IIADSILNLFGLGLIGPESP SVSAQNSDTP LLPPAVP human HDAC11 isoform 2 SEQ ID NO: 14MGLEKLHPFD AGKWGKVINF LKEEKLLSDS MLVEAREASEEDLLVVHTRR YLNELKRKVL RPLRTQTGGT IMAGKLAVERGWAINVGGGF HHCSSDRGGG FCAYADITLA IKFLFERVEGISRATIIDLD AHQGNGHERD FMDDKRVYIM DVYNRHIYPGDRFAKQAIRR KVELEWGTED DEYLDKVERN IKKSLQEHLPDVVVYNAGTD ILEGDRLGGL SISPAGIVKR DELVFRMVRGRRVPILMVTS GGYQKRTARI IADSILNLFG LGLIGPESPS VSAQNSDTPL LPPAVPhuman HDAC11 isoform 3 SEQ ID NO: 15MLHTTQLYQH VPETRWPIVY SPRYNITFMG LEKLHPFDAGKWGKVINFLK EEKLLSDSML VEAREASEED LLVVHTRRYLNELKFLFERV EGISRATIID LDAHQGNGHE RDFMDDKRVYIMDVYNRHIY PGDRFAKQAI RRKVELEWGT EDDEYLDKVERNIKKSLQEH LPDVVVYNAG TDILEGDRLG GLSISPAGIVKRDELVFRMV RGRRVPILMV TSGGYQKRTA RIIADSILNLFGLGLIGPES PSVSAQNSDT PLLPPAVP

HDAC11 Inhibitors

In certain embodiments, methods of the present disclosure directed tomodulating the activity of HDAC11 are practiced using an agent thatinhibits HDAC11 expression and/or activity. For example, in certainembodiments, such a modulator specifically reduces or inhibits HDAC11'sability to associate with a protein complex. In other embodiments, anagent reduces expression of HDAC11. In some embodiments, agents thatmodulate (e.g. inhibit) HDAC11 are polynucleotides, polypeptides,peptides, peptide nucleic acids, antibodies and fragments thereof, smallmolecules, inorganic compounds and/or organic compounds. In someembodiments, agents that modulate (e.g. inhibit) HDAC11 includeantagonists of HDAC11.

HDAC11 inhbitors include any molecule that reduces HDAC11 expression oractivity. In certain embodiments, inhibitors interfere with HDAC11activity by any of a variety of means including, e.g., inhibiting HDAC11associating with a protein complex or inhibiting deacetylase activity.

Any HDAC11 inhibitors known in the art may be used in the methodsdescribed herein. Examples of HDAC11 inhibitors include, but are notlimited to, HDTK010, HDTK028, HDTK029, HDTK054, HDTK070, commerciallyavailable shRNA (e.g., Sigma-Aldrich), commercially available siRNA(e.g., Sigma-Aldrich and Qiagen), commercially available Clusteredregular interspaced short palindromic repeats-CRISPR associated protein9-single guide RNA (CRISPR-Cas9-sgRNA) (e.g., Origene) and commerciallyavailable anti-HDAC11 antibodies (e.g., Abcam and Sigma-Aldrich).

In some embodiments, a HDAC11 inhibitor is a small molecule. In someembodiments, a small molecule HDAC11 inhibitor is at least 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold,2,000-fold, 3,000-fold, or more selective for inhibition of HDAC11 overone, two, three, four, five, six, seven, eight, or more other histonedeacetylase isoforms (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,HDAC7, HDAC8, HDAC9 and/or HDAC10). In certain embodiments, a HDAC11inhibitor is at least 10-fold selective for HDAC11 over other histonedeacetylase isoforms. In certain embodiments, the HDAC11 inhibitor is asmall molecule that is at least 200-fold selective for HDAC11 over otherhistone deacetylase isoforms. In some embodiments, a small moleculeHDAC11 inhibitor is at least 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold,300-fold, 400-fold, 500-fold, 1,000-fold, 2,000-fold, 3,000-fold, ormore selective for inhibition of HDAC11 each of HDAC1, HDAC2, HDAC3,HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9 and HDAC10.

Peptides and Polypeptides

In some embodiments, methods of the disclosure are practiced usingpeptide or polypeptide inhibitors of HDAC11. In some embodiments, aHDAC11 inhibitor is a peptide or polypeptide inhibitor of HDAC11.

In some embodiments, activity of HDAC11 is altered by over expression ofa dominant negative inhibitor of HDAC11. Dominant negative inhibitors ofHDAC11 are typically mutant forms of HDAC11, which reduce or block theactivity of wild type HDAC11, e.g., by competing for associating with aprotein complex or promoter but failing to deacetylate a target protein.Examples of various HDAC11 dominant negative inhibitors include a mutantHDAC11 having reduced ability to associate with a protein complex and amutant HDAC11 that associates with the protein complex but fails todeacetylate a target protein. For example, one dominant negative is aHDAC11 having one or more amino acid substitutions in the catalyticdomain, such that the mutant HDAC11 associates with the relevant proteincomplex but exhibits reduced deacetylation activity.

Polynucleotides

Various polynucleotides are contemplated for use as modulators of HDAC11expression and/or activity. In certain embodiments, polynucleotideinhibitors of HDAC11 are antisense RNA, or RNA interference (RNAi)reagents designed to specifically target HDAC11, according to methodsknown and available in the art. Other polynucleotide inhibitors include,e.g., targeting vectors designed for integration into the genome andsuitable for deleting all or a portion of a HDAC11 allele or mutating aHDAC11 allele, e.g., through insertional mutagenesis.

Antisense

In some embodiments, a HDAC11 inhibitor is an antisense RNA directed toHDAC11 polynucleotides, or other components of the HDAC11 signalingcascade. Antisense oligonucleotides have been demonstrated to beeffective and targeted inhibitors of protein synthesis, and,consequently, can be used to specifically inhibit protein synthesis by atargeted gene. The efficacy of antisense oligonucleotides for inhibitingprotein synthesis is well established. For example, antisense constructshave been described that inhibit and can be used to treat a variety ofabnormal cellular proliferations, e.g. cancer (U.S. Pat. No. 5,747,470;U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683).

The term “target sequence” refers to a portion of the target DNA or RNAagainst which the oligonucleotide or antisense agent is directed, thatis, the sequence to which the oligonucleotide will hybridize byWatson-Crick base pairing of a complementary sequence.

The term “targeting sequence” or “antisense targeting sequence” refersto the sequence in an oligonucleotide or other antisense agent that iscomplementary (meaning, in addition, substantially complementary) to thetarget sequence. The entire sequence, or only a portion, of theantisense compound may be complementary to the target sequence. Forexample, in an oligonucleotide having 20-30 bases, about 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,or 29 may be targeting sequences that are complementary to the targetregion. Typically, the targeting sequence is formed of contiguous bases,but may alternatively be formed of non-contiguous sequences that whenplaced together, e.g., from opposite ends of the oligonucleotide,constitute sequence that spans the target sequence.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, it may still be functionally “complementary.” Incertain embodiments, an oligonucleotide may have at most one mismatchwith the target sequence out of 10 nucleotides, and preferably at mostone mismatch out of 20. Alternatively, an oligonucleotide may have atleast 90% sequence homology, and preferably at least 95% sequencehomology, with the target sequence.

An oligonucleotide “specifically hybridizes” to a target polynucleotideif the oligomer hybridizes to the target under physiological conditions,with a Tm substantially greater than 45° C., preferably at least 50° C.,and typically 60° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions. At a given ionicstrength and pH, the Tm is the temperature at which 50% of a targetsequence hybridizes to a complementary polynucleotide. Again, suchhybridization may occur with “near” or “substantial” complementarity ofthe antisense oligomer to the target sequence, as well as with exactcomplementarity.

Therefore, in certain embodiments, oligonucleotide sequences thatcomprise all, or a portion of, any sequence that is capable ofspecifically binding to a HDAC11 target polynucleotide sequence, or acomplement thereof may be used in the methods described herein. Inanother embodiment, an oligonucleotide sequence comprises all, or aportion of, any sequence that is capable of specifically binding to aHDAC11 polynucleotide sequence, or a complement thereof. In someembodiments, an antisense oligonucleotides comprise DNA or derivativesthereof. In another embodiment, an oligonucleotides comprise RNA orderivatives thereof. Antisense oligonucleotides may be modified DNAscomprising a phosphorothioated modified backbone. Also, oligonucleotidesequences may comprise peptide nucleic acids or derivatives thereof. Ineach case, as appropriate, compositions can comprise a sequence regionthat is complementary to one or more portions of a HDAC11 target gene orpolynucleotide sequence. In some embodiments, compositions can comprisea sequence region that is completely complementary one or more portionsof a HDAC11 target gene or polynucleotide sequence.

Methods of producing antisense molecules are known in the art and can bereadily adapted to produce an antisense molecule that targets HDAC11.Selection of antisense compositions specific for a given sequence isbased upon analysis of the chosen target sequence and determination ofsecondary structure, Tm, binding energy, and relative stability.Antisense compositions may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware and/or the BLASTN 2.0.5 algorithm software (Altschul et al.,Nucleic Acids Res. 1997, 25(17):3389-402).

RNAi Molecules

RNA interference methods using RNAi molecules also may be used todisrupt the expression of a gene or polynucleotide of interest,including a HDAC11 gene. RNAi is an evolutionarily conservedgene-silencing mechanism, originally discovered in studies of thenematode Caenorhabditis elegans (Lee et al., Cell 75:843, 1993; Reinhartet al., Nature 403:901, 2000). It may be triggered by introducing dsRNAinto cells expressing the appropriate molecular machinery, which thendegrades the corresponding endogenous mRNA. Without wishing to be boundby theory, the RNAi mechanism involves conversion of dsRNA into shortRNAs that direct ribonucleases to homologous mRNA targets (summarized,for example, by Ruvkun, Science 2294:797, 2001).

In some embodiments, a HDAC11 inhibitor is a RNAi oligonucleotide. Insome embodiments a RNAi oligonucleotide is single stranded. In someembodiments, a RNAi oligonucleotide is double stranded.

In certain embodiments, methods provided herein may utilizedouble-stranded ribonucleic acid (dsRNA) molecules as modulating agents,for inhibiting HDAC11. dsRNAs generally comprise two single strands. Insome embodiments, one strand of the dsRNA comprises a nucleotidesequence that is substantially identical to a portion of the target geneor target region (the “sense” strand), and the other strand (the“complementary” or “antisense” strand) comprises a sequence that issubstantially complementary to a portion of the target region. Thestrands are sufficiently complementary to hybridize to form a duplexstructure. In certain embodiments, the complementary RNA strand may beless than 30 nucleotides, less than 25 nucleotides in length, or even 19to 24 nucleotides in length. In some embodiments, the complementarynucleotide sequence may be 20-23 nucleotides in length, or 22nucleotides in length.

In certain embodiments, at least one of the RNA strands comprises anucleotide overhang of 1 to 4 nucleotides in length. In otherembodiments, the dsRNA may further comprise at least one chemicallymodified nucleotide. In certain embodiments, a dsRNA comprising asingle-stranded overhang of 1 to 4 nucleotides may comprise a moleculewherein the unpaired nucleotide of the single-stranded overhang that isdirectly adjacent to the terminal nucleotide pair contains a purinebase. In some embodiments, the last complementary nucleotide pairs onboth ends of a dsRNA are a G-C pair, or, at least two of the last fourterminal nucleotide pairs are G-C pairs.

Certain embodiments of the present disclosure may comprise micro RNAs(miRNAs). In some embodiments, a HDAC11 inhibitor is a miRNA. miRNAsrepresent a large group of small RNAs produced naturally in organisms,some of which regulate the expression of target genes. miRNAs are formedfrom an approximately 70 nucleotide single-stranded hairpin precursortranscript by Dicer. (V. Ambros et al. Current Biology 13:807, 2003).miRNAs are not translated into proteins, but instead bind to specificmessenger RNAs, thereby blocking translation. Without wishing to bebound by theory, it is thought that miRNAs base-pair imprecisely withtheir targets to inhibit translation. Certain miRNAs may be transcribedas hairpin RNA precursors, which are then processed to their matureforms by Dicer enzyme.

Certain embodiments of the present disclosure may employshort-interfering RNAs (siRNA). In some embodiments, a HDAC11 inhibitoris a siRNA. In certain embodiments, the first strand of thedouble-stranded oligonucleotide contains two more nucleoside residuesthan the second strand. In other embodiments, the first strand and thesecond strand have the same number of nucleosides; however, the firstand second strands are offset such that the two terminal nucleosides onthe first and second strands are not paired with a residue on thecomplimentary strand. In certain instances, the two nucleosides that arenot paired are thymidine resides.

In instances where HDAC11 inhibiting agent is or comprises siRNA, theagent should include a region of sufficient homology to the targetregion, and be of sufficient length in terms of nucleotides, such thatthe siRNA agent, or a fragment thereof, can mediate down regulation ofthe target RNA. It will be understood that the term “ribonucleotide” or“nucleotide” can, in the case of a modified RNA or nucleotide surrogate,also refer to a modified nucleotide, or surrogate replacement moiety atone or more positions. Thus, a siRNA agent is or includes a region whichis at least partially complementary to the target RNA. It is notnecessary that there be perfect complementarity between the siRNA agentand the target, but the correspondence must be sufficient to enable thesiRNA agent, or a cleavage product thereof, to direct sequence specificsilencing, such as by RNAi cleavage of the target RNA. Complementarity,or degree of homology with the target strand, is most critical in theantisense strand. While perfect complementarity, particularly in theantisense strand, is often desired some embodiments include one or moremismatches with respect to the target RNA, typically 10, 8, 6, 5, 4, 3,2, or fewer mismatches. In some embodiments, matches are most toleratedin the terminal regions, and if present are preferably in a terminalregion or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5′and/or 3′ terminus. The sense strand need only be sufficientlycomplementary with the antisense strand to maintain the overalldouble-strand character of the molecule.

In addition, a siRNA modulating agent may be modified or includenucleoside surrogates. Single stranded regions of a siRNA agent may bemodified or include nucleoside surrogates, e.g., the unpaired region orregions of a hairpin structure, e.g., a region which links twocomplementary regions, can have modifications or nucleoside surrogates.Modification to stabilize one or more 3′- or 5′-terminus of a siRNAagent, e.g., against exonucleases, or to favor the antisense siRNA agentto enter into RISC are also useful. Modifications can include C3 (or C6,C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidicspacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethyleneglycol), special biotin or fluorescein reagents that come asphosphoramidites and that have another DMT-protected hydroxyl group,allowing multiple couplings during RNA synthesis.

siRNA agents may include, for example, molecules that are long enough totrigger the interferon response (which can be cleaved by Dicer(Bernstein et al., Nature, 409:363-366, 2001) and enter aRISC(RNAi-induced silencing complex)), in addition to molecules whichare sufficiently short that they do not trigger the interferon response(which molecules can also be cleaved by Dicer and/or enter a RISC),e.g., molecules which are of a size which allows entry into a RISC,e.g., molecules which resemble Dicer-cleavage products. Molecules thatare short enough that they do not trigger an interferon response aretermed siRNA agents or shorter RNAi agents herein. “siRNA agent orshorter RNAi agent” as used refers to an siRNA agent that issufficiently short that it does not induce a deleterious interferonresponse in a human cell, e.g., it has a duplexed region of less than 60but preferably less than 50, 40, or 30 nucleotide pairs. An siRNAmodulating agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA, suchas HDAC11 mRNA.

Each strand of an siRNA modulating agent can be equal to or less than35, 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand ispreferably at least 19 nucleotides in length. For example, each strandcan be between 21 and 25 nucleotides in length. Preferred siRNA agentshave a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotidepairs, and one or more overhangs, preferably one or two 3′ overhangs, of2-3 nucleotides.

In addition to homology to target RNA and the ability to down regulate atarget gene, an siRNA modulating agent may have one or more of thefollowing properties: it may, despite modifications, even to a verylarge number, or all of the nucleosides, have an antisense strand thatcan present bases (or modified bases) in the proper three dimensionalframework so as to be able to form correct base pairing and form aduplex structure with a homologous target RNA which is sufficient toallow down regulation of the target, e.g., by cleavage of the targetRNA; it may, despite modifications, even to a very large number, or allof the nucleosides, still have “RNA-like” properties, i.e., it maypossess the overall structural, chemical and physical properties of anRNA molecule, even though not exclusively, or even partly, ofribonucleotide-based content. For example, a siRNA agent can contain,e.g., a sense and/or an antisense strand in which all of the nucleotidesugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. Thisdeoxyribonucleotide-containing agent can still be expected to exhibitRNA-like properties. While not wishing to be bound by theory, theelectronegative fluorine prefers an axial orientation when attached tothe C2′ position of ribose. This spatial preference of fluorine can, inturn, force the sugars to adopt a C3′-endo pucker. This is the samepuckering mode as observed in RNA molecules and gives rise to theRNA-characteristic A-family-type helix. Further, since fluorine is agood hydrogen bond acceptor, it can participate in the same hydrogenbonding interactions with water molecules that are known to stabilizeRNA structures. Generally, it is preferred that a modified moiety at the2′ sugar position will be able to enter into H-bonding which is morecharacteristic of the OH moiety of a ribonucleotide than the H moiety ofa deoxyribonucleotide.

A “single strand RNAi agent” as used herein, is an RNAi agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand RNAi modulating agents arepreferably antisense with regard to the target molecule. A single strandRNAi agent should be sufficiently long that it can enter the RISC andparticipate in RISC mediated cleavage of a target mRNA. A single strandRNAi agent is at least 14, and more preferably at least 15, 20, 25, 29,35, 40, or 50 nucleotides in length. It is preferably less than 200,100, or 60 nucleotides in length.

Hairpin RNAi modulating agents, e.g., short hairpin RNA (shRNA), mayhave a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23,24, or 25 nucleotide pairs. The duplex region may preferably be equal toor less than 200, 100, or 50, in length. Certain ranges for the duplexregion are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs inlength. The hairpin may have a single strand overhang or terminalunpaired region, preferably the 3′, and preferably of the antisense sideof the hairpin. In certain embodiments, overhangs are 2-3 nucleotides inlength.

Certain modulating agents utilized according to the methods providedherein may comprise RNAi oligonucleotides such as chimericoligonucleotides, or “chimeras,” which contain two or more chemicallydistinct regions, each made up of at least one monomer unit, i.e., anucleotide in the case of an oligonucleotide compound. Theseoligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligonucleotides are used, compared tophosphorothioate oligodeoxynucleotides. Chimeric oligonucleotides may beformed as composite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleotides and/or oligonucleotide mimetics asdescribed above. Such oligonucleotides have also been referred to in theart as hybrids or gapmers. Representative United States patents thatteach the preparation of such hybrid structures include, but are notlimited to, U.S. Pat. Nos. 5,013,830, 5,149,797, 5,220,007, 5,256,775,5,366,878, 5,403,711, 5,491,133, 5,565,350, 5,623,065, 5,652,355,5,652,356, 5,700,922, and 5,955,589, each of which is hereinincorporated by reference. In certain embodiments, the chimericoligonucleotide is RNA-DNA, DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, orRNA-DNA-RNA-DNA, wherein the oligonucleotide is between 5 and 60nucleotides in length.

In some embodiments, HDAC11 modulating agents, such as RNAi agents,relate to an oligonucleotide comprising at least one ligand tethered toan altered or non-natural nucleobase. A large number of compounds canfunction as the altered base. The structure of the altered base isimportant to the extent that the altered base should not substantiallyprevent binding of the oligonucleotide to its target, e.g., mRNA. Incertain embodiments, the altered base is difluorotolyl, nitropyrrolyl,nitroimidazolyl, nitroindolyl, napthalenyl, anthrancenyl, pyridinyl,quinolinyl, pyrenyl, or the divalent radical of any one of thenon-natural nucleobases described herein. In certain embodiments, thenon-natural nucleobase is difluorotolyl, nitropyrrolyl, ornitroimidazolyl. In certain embodiments, a non-natural nucleobase isdifluorotolyl. A wide variety of ligands are known in the art and areamenable to the present invention. For example, a ligand can be asteroid, bile acid, lipid, folic acid, pyridoxal, B 12, riboflavin,biotin, aromatic compound, polycyclic compound, crown ether,intercalator, cleaver molecule, protein-binding agent, or carbohydrate.In certain embodiments, a ligand is a steroid or aromatic compound. Incertain instances, the ligand is cholesteryl.

In other embodiments, a RNAi agent is an oligonucleotide tethered to aligand for the purposes of improving cellular targeting and uptake. Forexample, a RNAi agent may be tethered to an antibody, or antigen bindingfragment thereof. As an additional example, an RNAi agent may betethered to a specific ligand binding molecule, such as a polypeptide orpolypeptide fragment that specifically binds a particular cell-surfacereceptor.

In some embodiments, a HDAC11 modulating agent comprises a non-naturalnucleobase. In certain embodiments, a non-natural nucleobase isdifluorotolyl, nitroimidazolyl, nitroindolyl, or nitropyrrolyl. Incertain embodiments, HDAC11 modulating agents provided herein relate toa double-stranded oligonucleotide sequence, wherein only one of the twostrands contains a non-natural nucleobase. In certain embodiments,HDAC11 modulating agents as used herein relate to a double-strandedoligonucleotide sequence, wherein both of the strands independentlycomprise at least one non-natural nucleobase.

In certain instances, a ribose sugar moiety that naturally occurs innucleosides is replaced with a hexose sugar. In some certainembodiments, a hexose sugar is an allose, altrose, glucose, mannose,gulose, idose, galactose, talose, or a derivative thereof. In somecertain embodiments, a hexose is a D-hexose. In certain instances, aribose sugar moiety that naturally occurs in nucleosides is replacedwith a polycyclic heteroalkyl ring or cyclohexenyl group. In certaininstances, a polycyclic heteroalkyl group is a bicyclic ring containingone oxygen atom in the ring. In certain instances, the polycyclicheteroalkyl group is a bicyclo[2.2.1]heptane, a bicyclo[3.2.1]octane, ora bicyclo[3.3.1]nonane. In certain embodiments, the backbone of anoligonucleotide has been modified to improve the therapeutic ordiagnostic properties of the oligonucleotide compound. In certainembodiments, at least one of the bases or at least one of the sugars ofthe oligonucleotide has been modified to improve the therapeutic ordiagnostic properties of the oligonucleotide compound. In instances whenthe oligonucleotide is double stranded, the two strands may becomplementary, partially complementary, or chimeric oligonucleotides.

Examples of modified RNAi agents envisioned for use in the methods ofthe present disclosure include oligonucleotides containing modifiedbackbones or non-natural internucleoside linkages. As defined here,oligonucleotides having modified backbones or internucleoside linkagesinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone. Modifiedoligonucleotides that do not have a phosphorus atom in their intersugarbackbone can also be considered to be oligonucleotides. Specificoligonucleotide chemical modifications are described below. It is notnecessary for all positions in a given compound to be uniformlymodified, and in fact more than one of the following modifications maybe incorporated in a single oligonucleotide compound or even in a singlenucleotide thereof.

Examples of modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalklyphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States patents that teach the preparation of theabove phosphorus atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243,5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717,5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677,5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253,5,571,799, 5,587,361, 5,625,050, and 5,697,248, each of which is hereinincorporated by reference.

Examples of modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleotides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleotides include, but are not limited to, U.S. Pat. Nos.5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033,5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967,5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,610,289,5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312,5,633,360, 5,677,437, and 5,677,439, each of which is hereinincorporated by reference.

In other examples of oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleoside units maybe replaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigonucleotide, an oligonucleotide mimetic, that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide-containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082,5,714,331, and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found in Nielsen etal., Science, 1991, 254, 1497.

In certain instances, the RNAi agents for use with the methods providedherein may be modified by non-ligand group. A number of non-ligandmolecules have been conjugated to oligonucleotides in order to enhancethe activity, cellular distribution, cellular targeting, or cellularuptake of the oligonucleotide, and procedures for performing suchconjugations are available in the scientific literature. Such non-ligandmoieties have included lipid moieties, such as cholesterol (Letsinger etal., Proc. Natl. Acad. Sci. USA, 86:6553-56, 1989), cholic acid(Manoharan et al., Bioorg. Med. Chem. Lett. 4:1053, 1994), a thioether,e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,660:306, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765, 1993),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 20:533, 1992),an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J. 10:111, 1991; Kabanov et al., FEBSLett. 259:327, 1990; Svinarchuk et al., Biochimie. 75:49, 1993), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 36:3651, 1995; Shea et al., Nucl. Acids Res. 18:3777,1990), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides. 14:969, 1995), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett. 36:3651, 1995), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta. 1264:229, 1995), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther. 277:923, 1996). Representative UnitedStates patents that teach the preparation of such oligonucleotideconjugates have been listed above. Typical conjugation protocols involvethe synthesis of oligonucleotides bearing an aminolinker at one or morepositions of the sequence. The amino group is then reacted with themolecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with theoligonucleotide still bound to the solid support or following cleavageof the oligonucleotide in solution phase. Purification of theoligonucleotide conjugate by HPLC typically affords the pure conjugate.

Additional examples of modulating agents, such as RNAi oligonucleotides,may be found in U.S. Application Publication Nos. 2007/0275465,2007/0054279, 2006/0287260, 2006/0035254, 2006/0008822, which areincorporated by reference.

CRISPR

In some embodiments, a gene editing system can be used to silence,enhance or mutate the HDAC11 gene. Any gene editing systems known in theart may be used in the methods of the present disclosure. In certainembodiments, a gene editing system is a CRISPR/Cas system. CRISPR/Cassystems have been modified for use in gene editing (silencing, enhancingor changing specific genes) in eukaryotes such as mice or primates.Wiedenheft et al., 2012. Nature 482: 331-8. This is accomplished byintroducing into the eukaryotic cell a plasmid containing a specificallydesigned CRISPR and one or more appropriate Cas. The CRISPR sequence,sometimes called a CRISPR locus, comprises alternating repeats andspacers. In a naturally-occurring CRISPR, the spacers usually comprisesequences foreign to the bacterium such as a plasmid or phage sequence;in the HDAC11 CRISPR/Cas system, the spacers are derived from the HDAC11gene sequence. RNA from the CRISPR locus is constitutively expressed andprocessed by Cas proteins into small RNAs. These comprise a spacerflanked by a repeat sequence. The RNAs guide other Cas proteins tosilence exogenous genetic elements at the RNA or DNA leval. Horvath etal. 2010. Science 327: 167-170; Makarova et al. 2006 Biology Direct 1:7.The spacers thus serve as templates for RNA molecules, analogously tosiRNAs. Pennisi 2013. Science 341: 833-836.

In certain embodiments, artificial CRISPR systems can be generated whichinhibit HDAC11 using technology known in the art, e.g., that isdescribed in U.S. patent application Ser. No. 13/842,859 (published asUS 20140068797). Such HDAC11-inhibitory CRISPR system can include aguide RNA (gRNA) comprising a HDAC11-targeting domain, i.e, a nucleotidesequence that is complementary to a HDAC11 DNA strand, and a seconddomain that interacts with an RNA-directed nuclease.

Knockout Constructs

In certain embodiments, the activity of HDAC11 is altered by mutating agene encoding the HDAC11 molecule. A variety of methods of mutating anendogenous gene are known and available in the art, including, e.g.,insertional mutagenesis and knockout methods. Accordingly, thedisclosure provides methods of knocking out one or more alleles of aHDAC11 gene. It is understood that knockout vectors in accordance withthe present disclosure include any vector capable of disruptingexpression or activity of a HDAC11 gene, including, in certainembodiments, targeting vectors.

In preferred methods, targeting vectors are used to selectively disrupta HDAC11 gene. Knockout vectors include those that alter geneexpression, for example, by disrupting a regulatory element of a HDAC11gene, including, e.g., inserting a regulatory element that reduces geneexpression or deleting or otherwise reducing the activity of anendogenous element that positively affects transcription of the targetgene. In other embodiments, knockout vectors can disrupt, e.g., deleteor mutate, the 5′ region, 3′ region or coding region of a HDAC11 gene.In some embodiments, knockout vectors delete a region or the entirety ofthe coding region of a HDAC11 gene. In certain embodiments, knockoutvectors delete a region of a HDAC11 gene, while in other embodiments;they insert exogenous sequences into a HDAC11 gene. In addition, incertain embodiments, including those using replacement vectors, knockoutvectors both remove a region of a gene and introduce an exogenoussequence.

Targeting vectors in accordance with the present disclosure include allvectors capable of undergoing homologous recombination with anendogenous HDAC11 gene, including replacement vectors. Targeting vectorsinclude all those used in methods of positive selection, negativeselection, positive-negative selection, and positive switch selection.Targeting vectors employing positive, negative, and positive-negativeselection are well known in the art and representative examples aredescribed in Joyner, A. L., Gene Targeting: A Practical Approach, 2ndEd. (2000) and references cited therein.

Antibody Agents

In some embodiments, antibody agents that specifically bind HDAC11 canbe used in accordance with the methods described herein. In someembodiments, an antibody agent that specifically binds to HDAC11 andinhibits its catalytic activity. As used herein, the term “antibodyagent” refers to an agent that specifically binds to a particularantigen. In some embodiments, the term encompasses any polypeptide orpolypeptide complex that includes immunoglobulin structural elementssufficient to confer specific binding. Exemplary antibody agentsinclude, but are not limited to monoclonal antibodies or polyclonalantibodies. In some embodiments, an antibody agent may include one ormore constant region sequences that are characteristic of mouse, rabbit,primate, or human antibodies. In some embodiments, an antibody agent mayinclude one or more sequence elements are humanized, primatized,chimeric, etc, as is known in the art. In many embodiments, the term“antibody agent” is used to refer to one or more of the art-known ordeveloped constructs or formats for utilizing antibody structural andfunctional features in alternative presentation. For example,embodiments, an antibody agent utilized in accordance with the presentdisclosure is in a format selected from, but not limited to, intact IgA,IgG, IgE or IgM antibodies; bi- or multi-specific antibodies (e.g.,Zybodies®, etc); antibody fragments such as Fab fragments, Fab′fragments, F(ab′)2 fragments, Fd′ fragments, Fd fragments, and isolatedCDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; singledomain antibodies (e.g., shark single domain antibodies such as IgNAR orfragments thereof); cameloid antibodies; masked antibodies (e.g.,Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); singlechain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies®minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®;DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®;Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; andKALBITOR®s. In some embodiments, an antibody may lack a covalentmodification (e.g., attachment of a glycan) that it would have ifproduced naturally. In some embodiments, an antibody may contain acovalent modification (e.g., attachment of a glycan, a payload [e.g., adetectable moiety, a therapeutic moiety, a catalytic moiety, etc], orother pendant group [e.g., poly-ethylene glycol, etc.]. In manyembodiments, an antibody agent is or comprises a polypeptide whose aminoacid sequence includes one or more structural elements recognized bythose skilled in the art as a complementarity determining region (CDR);in some embodiments an antibody agent is or comprises a polypeptidewhose amino acid sequence includes at least one CDR (e.g., at least oneheavy chain CDR and/or at least one light chain CDR) that issubstantially identical to one found in a reference antibody. In someembodiments an included CDR is substantially identical to a referenceCDR in that it is either identical in sequence or contains between 1-5amino acid substitutions as compared with the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with thereference CDR. In some embodiments an included CDR is substantiallyidentical to a reference CDR in that it shows at least 96%, 96%, 97%,98%, 99%, or 100% sequence identity with the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that at least one amino acid within the included CDR is deleted,added, or substituted as compared with the reference CDR but theincluded CDR has an amino acid sequence that is otherwise identical withthat of the reference CDR. In some embodiments an included CDR issubstantially identical to a reference CDR in that 1-5 amino acidswithin the included CDR are deleted, added, or substituted as comparedwith the reference CDR but the included CDR has an amino acid sequencethat is otherwise identical to the reference CDR. In some embodiments anincluded CDR is substantially identical to a reference CDR in that atleast one amino acid within the included CDR is substituted as comparedwith the reference CDR but the included CDR has an amino acid sequencethat is otherwise identical with that of the reference CDR. In someembodiments an included CDR is substantially identical to a referenceCDR in that 1-5 amino acids within the included CDR are deleted, added,or substituted as compared with the reference CDR but the included CDRhas an amino acid sequence that is otherwise identical to the referenceCDR. In some embodiments, an antibody agent is or comprises apolypeptide whose amino acid sequence includes structural elementsrecognized by those skilled in the art as an immunoglobulin variabledomain. In some embodiments, an antibody agent is a polypeptide proteinhaving a binding domain which is homologous or largely homologous to animmunoglobulin-binding domain.

An antibody agent is said to “specifically bind,” “immunologicallybind,” and/or is “immunologically reactive” to a polypeptide of thedisclosure if it reacts at a detectable level (within, for example, anELISA assay) with the polypeptide, and does not react detectably withunrelated polypeptides under similar conditions. Antibodies areconsidered to specifically bind to a target polypeptide when the bindingaffinity is at least 1×10⁻⁷ M or, preferably, at least 1×10⁻⁸ M. In someembodiments, a HDAC11 inhibitory agent is an antibody agent thatspecifically binds the catalytic domain of HDAC11.

Antibody agents for use in accordance with the present disclosureinclude, but are not limited to, monoclonal antibodies, chimericantibodies, humanized antibodies, fully human antibodies, single chainantibodies, Fab fragments, and scFv fragments. An example of ananti-HDAC11 antibody is described in WO 2014/096386.

Antibody agents may be prepared by any of a variety of techniques knownto those of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Ingeneral, antibodies can be produced by cell culture techniques,including the generation of monoclonal antibodies via conventionaltechniques known in the art, or via transfection of antibody genes intosuitable bacterial or mammalian cell hosts, in order to allow for theproduction of recombinant antibodies. Monoclonal antibodies (Mabs)specific for an antigenic polypeptide of interest may be prepared, forexample, using the technique of Kohler and Milstein, Eur. J. Immunol.6:511-519, 1976, and improvements thereto. Methods of making chimericand humanized antibodies are well known in the art, (See, e.g., U.S.Pat. No. 4,816,567, International Application No. WO84/03712,respectively).

In certain embodiments, methods of preparing monoclonal antibodiesinvolve the preparation of immortal cell lines capable of producingantibodies having the desired specificity (i.e., reactivity with thepolypeptide of interest). Such cell lines may be produced, for example,from spleen cells obtained from an animal immunized as described above.The spleen cells are then immortalized by, for example, fusion with amyeloma cell fusion partner, preferably one that is syngeneic with theimmunized animal. A variety of fusion techniques may be employed. Forexample, the spleen cells and myeloma cells may be combined with anonionic detergent for a few minutes and then plated at low density on aselective medium that supports the growth of hybrid cells, but notmyeloma cells. A preferred selection technique uses HAT (hypoxanthine,aminopterin, thymidine) selection. After a sufficient time, usuallyabout 1 to 2 weeks, colonies of hybrids are observed. Single coloniesare selected and their culture supernatants tested for binding activityagainst the polypeptide. Hybridomas having high reactivity andspecificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides in accordance with thepresent disclosure may be used in the purification process in, forexample, an affinity chromatography step.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)2” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent VH::VL heterodimer including an antigen-bindingsite which retains much of the antigen recognition and bindingcapabilities of the native antibody molecule. Inbar et al. (1972) Proc.Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VLheterodimer which is expressed from a gene fusion including VH- andVL-encoding genes linked by a peptide-encoding linker. Huston et al.(1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methodshave been described to discern chemical structures for converting thenaturally aggregated—but chemically separated—light and heavypolypeptide chains from an antibody V region into an scFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

In certain embodiments, each of the above-described antibody agents caninclude a heavy chain and a light chain complementarity-determiningregion (CDR) set, respectively interposed between a heavy chain and alight chain framework region which provide support to the CDRS anddefine the spatial relationship of the CDRs relative to each other. Asused herein, the term “CDR set” refers to the three hypervariableregions of a heavy or light chain V region. Proceeding from theN-terminus of a heavy or light chain, these regions are denoted as“CDR1,” “CDR2,” and “CDR3” respectively. An antigen-binding site,therefore, includes six CDRs, comprising the CDR set from each of aheavy and a light chain V region. A polypeptide comprising a single CDR,(e.g., a CDR1, CDR2 or CDR3) is referred to herein as a “molecularrecognition unit.” Crystallographic analysis of a number ofantigen-antibody complexes has demonstrated that the amino acid residuesof CDRs form extensive contact with bound antigen, wherein the mostextensive antigen contact is with the heavy chain CDR3. Thus, themolecular recognition units are primarily responsible for thespecificity of an antigen-binding site.

Fab or F(ab′)2 fragments may be wholly animal or human derived, or theymay be in chimeric form, such that the constant domains are derived fromthe constant regions of human immunoglobulins and the variable regionsare derived from the parent murine mAb. Alternatively, the Fv, Fab, orF(ab′)2 may be humanized, so that only the complementarity determiningregions (CDR) are derived from an animal mAb, and the constant domainsand the framework regions of the variable regions are of human origin.These chimeric and humanized fragments are less immunogenic than theirwholly animal counterparts, and thus more suitable for in vivo use,especially over prolonged periods.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is may be accepted asthe human framework region (FR) for the humanized antibody (Sims et al.,J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

Humanization generally aims to retain high affinity for the antigen ofinterest and other favorable biological properties. To achieve thisgoal, humanized antibodies may be prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and5,545,807.

Humanized or fully human antibodies in accordance with the presentdisclosure may be prepared according to the methods described in U.S.Pat. Nos. 5,770,429, 5,833,985, 5,837,243, 5,922,845, 6,071,517,6,096,311, 6,111,166, 6,270,765, 6,303,755, 6,365,116, 6,410,690,6,682,928, and 6,984,720.

Phage display technology (McCafferty et al., Nature 348:552-553 (1990))can also be used to produce human antibodies and antibody fragments invitro, from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B cell.

Phage display can be performed in a variety of formats; for their reviewsee, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion inStructural Biology 3:564-571 (1993). Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature, 352:624-628(1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991),or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos.5,565,332 and 5,573,905.

Phage display may be used to generate synthetic antibodies or humanantibodies according to various methods. For example, in certainembodiments, phage display is utilized to produce a library of fullyhuman antibodies or fragments thereof, which may be screened for theirability to bind a target polypeptide, such as HDAC11. The screening ofsuch libraries is commercially available, e.g., by Morphosys (Munich,Germany). Libraries of human antibodies and methods of use thereof aredescribed, e.g., in U.S. Pat. Nos. 7,049,135, 6,828,422, 6,753,138,6,706, and 484, 6,696,248, and U.S. Patent Application Publication Nos.2006/0121563, 2006/0003334, and 2004/0157291, all assigned to Morphosys.

Additional methods of generating and screening human antibody librariesusing phage display are described, e.g., in Steukers, M. et al., JImmunol Methods. 2006 Mar. 20; 310(1-2):126-35; Huang L. et al., J.Leukoc. Biol. Vol 80. 2006 October; Wassaf, D. et al., Anal Biochem.2006 Apr. 15; 351(2):241-53; Shrivastava A, et al., Protein Eng Des Sel.2005 September; 18(9):417-24; Schoonbroodt S. et al., Nucleic Acids Res.2005 May 19; 33(9); Hogan S, et al., Biotechniques. 2005 April;38(4):536, 538; Hoet R M, et al., Nat Biotechnol. 2005 March;23(3):344-8; Huang L, et al., J Mol Recognit. 2005 February 10; BlaiseL, et al., Gene. 2004 Nov. 24; 342(2):211-8; Fleming T, et al., J. Mol.Recognit. 2004 17:1-9; Jostock, et al., J Immunol Methods. 2004 June;289(1-2):65-80; Kelley B, et al., J Chro. 4 Jun. 2004 2004;1038(1-2):121-130; Ladner R C, et al., Drug Discovery Today. June 2004;9(12):525-529; Williams A, Baird L G, Transfus Apheresis Sci. December2003; 29(3):255-258; van den Beucken T, et al., FEBS Lett. Jul. 10,2003; 546(2-3):288-294. Jul. 10, 2003; 546(2-3):288-294; Sato A.,Biopolymers. July 2003; 71(3):316; and Nixon A E., Biopolymers. July2003; 71(3):302, 398.

In some embodiments, an antibody or fragment thereof inhibits HDAC11activity by binding to HDAC11 and, e.g., blocking the catalytic domain,and thereby inhibiting the deacetylase activity.

Small Molecules

In some embodiments, HDAC11 inhibitors for use in accordance with thepresent disclosure are chemical compounds, including large or smallinorganic or organic molecules. In some embodiments, HDAC11 modulatingagents in accordance with the present disclosure are small organicmolecules, or derivatives or analogs thereof.

In certain embodiments, a HDAC11 modulating agent includes a protectinggroup. The term “protecting group” refers to chemical moieties thatblock at least some reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved (or “cleaved”). Examples of blocking/protecting groups aredescribed, e.g., in Greene and Wuts, Protective Groups in OrganicSynthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999.

Any of the HDAC11-inhibiting chemical compounds may possess one or morechiral centers and each center may exist in the R or S configuration. Insome embodiments, a chiral center exists in the R configuration. In someembodiments, a chiral center exists in the S configuration.HDAC11-inhibiting chemical compounds of the present disclosure includeall diastereomeric, enantiomeric, and epimeric forms as well as mixturesthereof. Stereoisomers may be obtained, if desired, by methods known inthe art as, for example, the separation of stereoisomers by chiralchromatographic columns. HDAC11-inhibiting chemical compounds mayfurther include of N-oxides, crystalline forms (also known aspolymorphs), and pharmaceutically acceptable salts, as well as activemetabolites of any inhibitor. All tautomers are included within thescope of the HDAC11 modulating agents presented herein. In addition,HDAC11-inhibiting chemical compounds agents described herein can existin unsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. The solvated forms ofHDAC11-inhibiting chemical compounds are also included within thepresent disclosure.

In some embodiments, a small molecule inhibitor binds to HDAC11. In someembodiments, a small molecule binds to the catalytic domain of HDAC11and interferes with or reduces its deacetylase activity or its abilityto associate with other proteins to form a complex. In some embodiments,a small molecule HDAC11 inhibitor is at least 10-fold selective for theinhibition of HDAC11 over one or more other histone deacetylase isoforms(e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9and/or HDAC10). In other embodiments, a small molecule inhibitor ofHDAC11 is at least 200-fold selective for HDAC11 over other isoforms ofhistone deacetylases. In some embodiments, a small molecule HDAC11inhibitor is at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold,400-fold, 500-fold, 1,000-fold, 2,000-fold, 3,000-fold, or moreselective for inhibition of HDAC11 over one or more other histonedeacetylase isoforms (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,HDAC7, HDAC8, HDAC9 and/or HDAC10). In some embodiments, a smallmolecule HDAC11 inhibitor is at least 10-fold selective for theinhibition of HDAC11 over each of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,HDAC6, HDAC7, HDAC8, HDAC9 and HDAC10. In some embodiments, a HDAC11inhibitor is specific for huma HDAC11.

Inhibitors of HDAC11, including small organic compounds, may beidentified according to routine screening procedures available in theart, e.g., using commercially available libraries of such compounds.Exemplary inhibitors of HDAC11 are described in further detail below.

Aminobenzimidazoles and Related Compounds

In one embodiment of the invention, compounds of Formula I aredescribed:

and pharmaceutically acceptable salts thereof wherein:

Q is —H, —OC(O)NR⁶(C₁-C₆)alkylaryl, or —OC(O)O(C₁-C₆)alkylaryl;

Z is —CH₂—, O, S or NR⁶;

X₁, X₂, X₃, and X₄ are each independently, at each occurrence, N or CR¹;

Y₁, Y₂, and Y₃ are each independently N or CR¹;

L is NR⁶, O, or —(CR¹R²)_(p)—;

R¹ and R² are independently, at each occurrence, —H, —R³, —R⁴,—C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,—C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5heteroatoms selected from the group consisting of N, S, P and O, —OH,—OR³, halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂, —S(O)₂NH₂,—S(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)₂R⁵, —S(O)₂(C₁-C₆alkyl),—(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,—N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, or —(CHR⁵)_(p)NR³R⁴, wherein each alkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with one or more —OH, halogen,—NO₂, oxo, —CN, —R³, —R⁵, —SR³, —OR³, —NHR³, —NR³R⁴, —S(O)₂N(R³)₂—,—S(O)₂R⁵, —C(O)R⁵, —C(O)OR⁵, —NR³S(O)₂R⁵, —S(O)R⁵, —S(O)NR³R⁴,—NR³S(O)R⁵, heterocycle, aryl, or heteroaryl;

or R¹ and R² can combine with the carbon atom to which they are bothattached to form a spirocycle, spiroheterocycle, or spirocycloalkenyl,each optionally substituted with one or more independent occurrences ofR³ and R⁴;

or two occurrences of R¹, when on adjacent atoms, can combine to form acycloalkyl, a heterocycle, aryl, heteroaryl containing 1-5 heteroatomsselected from the group consisting of N, S, P and O, or a cycloalkenyl,each optionally substituted with one or more independent occurrences ofR³ and R⁴;

R³ and R⁴ are independently, at each occurrence, —H, —C₁-C₆alkyl,—C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl,heterocyclyl, aryl, heteroaryl containing 1-5 heteroatoms selected fromN, S, P, and O, —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂(C₁-C₆alkyl),—(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, oxo, or—(CHR⁵)_(p)N(C₁-C₆alkyl)₂, wherein each alkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionallysubstituted with one or more substituents selected from —OH, halogen,—NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl, —N(C₁-C₆alkly)₂,—S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl,—C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵,—S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, orheteroaryl;

R⁵ is independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆alkenyl,—C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,heteroaryl containing 1-5 heteroatoms selected from N, S, P and O, —OH,halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,—S(O)₂NH(C₁-C₆alkyl), —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂C₁-C₆alkyl,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)SO₂C₁-C₆alkyl,—S(O)(C₁-C₆alkyl), —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)(C₁-C₆alkyl)or —(CH₂)_(p)N(C₁-C₆alkyl)₂;

R⁶ is independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆alkenyl,—C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,heteroaryl containing 1-5 heteroatoms selected from the group consistingof N, S, P and O, —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂(C₁-C₆alkyl),—(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, or—(CHR⁵)_(p)NR³R⁴ wherein each alkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith one or more substituents selected from —OH, halogen, —NO₂, oxo,—CN, —R⁵, —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl, —N(C₁-C₆alkyl)₂,—S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl,—C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵,—S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, orheteroaryl;

and

p is 0, 1, 2, 3, 4, 5, or 6.

In some embodiments, compounds of Formula I′ are described:

wherein Y₄ is N or CR¹, and all other variables are as defined above forFormula I and described in classes and subclasses herein. Unlessotherwise stated, references herein to Formula I include reference toFormula I′.

In some embodiments, for compounds of Formula I, one of Y₁, Y₂ or Y₃ isN and the other two of Y₁, Y₂ or Y₃ are each independently CR¹. In otherembodiments, two of Y₁, Y₂ or Y₃ are N and the other one of Y₁, Y₂ or Y₃is CR¹. In some embodiments, R¹ is H.

In some embodiments, Y₁, Y₂ and Y₃ are CH; X₁, X₂, X₃ and X₄ are eachCR¹; and R¹ is independently, at each occurrence, —H, —R³, —R⁴,—C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,—C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5heteroatoms selected from the group consisting of N, S, P and O, —OH,halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,—S(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)₂R⁵, —S(O)₂(C1-C₆alkyl),—(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,—N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, or —(CHR⁵)_(p)NR³R⁴, wherein each alkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with one or more —OH, halogen,—NO₂, oxo, —CN, —R⁵, —OR³, —NHR³, —NR³R⁴, —S(O)₂N(R³)₂—, —S(O)₂R⁵,—C(O)R⁵, —C(O)OR⁵, —NR³S(O)₂R⁵, —S(O)R⁵, —S(O)NR³R⁴, —NR³S(O)R⁵,heterocycle, aryl, or heteroaryl;

or R¹ and R² can combine with the carbon atom to which they are bothattached to form a spirocycle, spiroheterocycle, or spirocycloalkenyl,each optionally substituted with one or more independent occurrences ofR³ and R⁴;

or two occurrences of R¹, when on adjacent atoms, can combine to form acycloalkyl, a heterocycle, aryl, heteroaryl containing 1-5 heteroatomsselected from the group consisting of N, S, P and O, or a cycloalkenyl,each optionally substituted with one or more independent occurrences ofR³ and R⁴;

R³ and R⁴ are independently, at each occurrence, —H, —C₁-C₆alkyl,—C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl,heterocyclyl, aryl, heteroaryl containing 1-5 heteroatoms selected fromN, S, P, and O, —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂(C₁-C₆alkyl),—(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, or—(CHR⁵)_(p)N(C₁-C₆alkyl)₂, wherein each alkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionallysubstituted with one or more substituents selected from —OH, halogen,—NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl, —N(C₁-C₆alkly)₂,—S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl,—C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵,—S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, orheteroaryl;

R⁵ is independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆alkenyl,—C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,heteroaryl containing 1-5 heteroatoms selected from N, S, P and O, —OH,halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,—S(O)₂NH(C₁-C₆alkyl), —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂C₁-C₆alkyl,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)SO₂C₁-C₆alkyl,—S(O)(C₁-C₆alkyl), —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)(C₁-C₆alkyl)or —(CH₂)_(p)N(C₁-C₆alkyl)₂.

In some embodiments, for compounds of Formula I, L is S. In otherembodiments, for compounds of Formula I, L is O. In other embodimentsfor compounds of Formula I, L is NR⁶.

In one or more embodiments, compounds of Formula I-A are provided:

wherein X₁, X₂, X₃, X₄, and R⁶ are as described generally above and inclasses, subclasses, and species herein.

In one or more embodiments, compounds of Formula I-B are provided:

wherein X₁, X₂, X₃, X₄, and R⁶ are as described generally above and inclasses, subclasses, and species herein.

In one or more embodiments, compounds of Formula I-C are provided:

wherein X₁, X₂, X₃, X₄, and R⁶ are as described generally above and inclasses, subclasses, and species herein.

In some embodiments for compounds of Formulae I, I-A, I-B, or I-C, X₁and X₄ are both N, and X₂ and X₃ are each independently CR¹. In someembodiments for compounds of Formulae I, I-A, I-B, or I-C, X₂ and X₄ areboth N, and X₁ and X₃ are each independently CR¹. In some embodimentsfor compounds of Formulae I, I-A, I-B, or I-C, X₁ is N and X₂, X₃ and X₄are each independently CR¹.

In some embodiments for compounds of Formulae I, I-A, I-B, or I-C, eachoccurrence of R⁶ is independently H, —C₁-C₆alkyl, —(C₁-C₆alkyl)S(O)₂R⁵,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, or —(CHR⁵)_(p)NR³R⁴ wherein eachalkyl is optionally substituted with one or more substituents selectedfrom —OH, halogen, —NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl,—N(C₁-C₆alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl,—S(O)R⁵, —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl,or heteroaryl. In some embodiments, for compounds of Formulae I, I-A,I-B, or I-C, each occurrence of R⁶ is independently H, —(CHR⁵)_(p)NR³R⁴,or —C₁-C₆alkyl optionally substituted with one or more substituentsselected from —OH, halogen, —NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl,—NH(C₁-C₆)alkyl, —N(C₁-C₆alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂,—S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,—N(C₁-C₆alkyl)S(O)₂C1-C₆alkyl, —S(O)R⁵, —S(O)N(C₁-C₆alkyl)₂, or—N(C₁-C₆alkyl)S(O)R⁵.

For each of the embodiments described above for compounds of Formulae I,I-A, I-B, or I-C, each independent occurrence of R¹ is halogen, —CF₃,—OH, —CN, —SO₂(C₁-C₃alkyl), phenyl, C₁-C₃alkyl, C₁-C₃alkoxy, pyridyl,—C(O)C₁-C₃alkyl, —OC₁-C₃alkyl, —(C₁-C₃alkyl)O(C1-C₃alkyl), —OCF₃ or—OCH₂phenyl.

In some embodiments, in any of the above-embodiments for compounds ofFormula I, I-A, I-B, or I-C, Q is —H.

In one or more embodiments, a compound of Formula I can be selected fromone of the compounds in Table I-1:

TABLE I-1 Example Structure Name HDTK010

3-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-N-hydroxybenzamide 2-1

3-((6-cyano-5- (trifluoromethyl)benzo[d]oxazol-2-yl)amino)-N-hydroxybenzamide 3-1

N-hydroxy-3-((6-phenyl-5- (trifluorornethyl)benzo[d]oxazol-2-yl)amino)benzamide 4-1

5-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-2-fluoro-N-hydroxy-benzamide 4-2

3-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-2-fluoro-N-hydroxy-benzamide 4-3

3-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-4-fluoro-N-hydroxy-benzamide 4-4

3-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-5-fluoro-N-hydroxy-benzamide 4-5

5-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-N-hydroxynicotinamide 4-6

4-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-N-hydroxypicolinamide 4-7

2-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-N-hydroxyisonicotinamide 4-8

2-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-N-hydroxypyrimidine-4-carboxamide 5-1

3-((5,6-dichlorobenzo[d]thiazol-2- yl)amino)-N-hydroxybenzamide 6-1

3-((6-cyano-5-(methylsulfonyl)-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 7-1

N-hydroxy-3-((5-(methylsulfonyl)-6-(trifluoromethyl)-1H-benzo[d]imidazol- 2-yl)amino)benzamide 8-1

3-((1H-benzo[d]imidazol-2-yl)amino)- N-hydroxybenzamide 9-1

3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)(methyl)amino)- N-hydroxybenzamide 10-1 

3-((5-cyano-1-methyl-6-(trifluoro- methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 11-1 

3-((6-cyano-1-methyl-5-(trifluoro- methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 12-1 

3-((6-cyano-5-fluoro-1-methyl-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 13-1 

3-((5,6-dichloro-1-methyl-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 14-1 

3-((6-cyano-1-isopropyl-5- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 15-1 

3-((5-cyano-1-isopropyl-6- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 16-1 

N-hydroxy-3-((1-isopropyl-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol- 2-yl)amino)benzamide 16-2 

N-hydroxy-3-((1-isopropyl-5-phenyl-6-(trifluoromethyl)-1H-benzo[d]imidazol- 2-yl)amino)benzamide 17-1 

3-((5,6-dichloro-1-isopropyl-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 18-1 

3-((6-cyano-1-(2-methoxyethyl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 19-1 

3-((5-cyano-1-(2-methoxyethyl)-6- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 20-1 

N-hydroxy-3-((1-(2-methoxyethyl)-6- phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 20-2 

N-hydroxy-3-((1-(2-methoxyethyl)-5- phenyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 21-1 

3-((1-(5-aminopentyl)-5-cyano-6- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 22-1 

3-((1-(5-aminopentyl)-5,6-dichloro- 1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 22-2 

3-((1-(5-aminopentyl)-5-chloro-6- fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 23-1 

N-hydroxy-3-((1-(2-methoxyethyl)- 1H-benzo[d]imidazol-2-yl)amino)benzamide 23-2 

3-((1H-benzo[d]imidazol-2-yl)amino)- N-hydroxy-1-naphthamide 23-3 

N-hydroxy-3-((1-(2-methoxyethyl)- 1H-benzo[d]imidazol-2-yl)amino)-1-naphthamide 23-4 

3-((5,6-dimethyl-1H-benzo[d]imidazol- 2-yl)amino)-N-hydroxybenzamide24-1 

N-hydroxy-3-((4-methoxy-1-(2- methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide 24-2 

N-hydroxy-3-((7-methoxy-1-(2- methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide 25-1 

3-((1H-benzo[d]imidazol-2-yl)(2- methoxyethyl)amino)-N-hydroxy-benzamide 26-1 

3-((5-cyano-6-(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)oxy)-N-hydroxybenzamide 27-1 

3-((5-cyano-6-(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-N-hydroxybenzamide 27-2 

3-((5,6-dichloro-1H-benzo[d]imidazol- 2-yl)methyl)-N-hydroxybenzamide28-1 

3-((5,6-dichloro-1H-indol-2-yl)amino)- N-hydroxybenzamide 29-1 

3-((1H-imidazo[4,5-b]pyrazin-2-yl) amino)-N-hydroxybenzamide 30-1 

3-((9H-purin-8-yl)amino)-N- hydroxybenzamide 31-1 

6-((5,6-dichlorobenzo[d]oxazol-2- yl)amino)-N-hydroxypicolinamide 32-1 

N-hydroxy-3-(oxazolo[4,5-b]pyridin- 2-ylamino)benzamide 32-2 

N-hydroxy-3-((6- (trifluoromethyl)oxazolo[4,5-b]pyridin-2-yl)amino)benzamide 33-1 

3-((6-chloro-5-fluoro-1H-benzo[d] imidazol-2-yl)amino)-N-hydroxy-benzamide 33-10

3-((5-fluoro-6-methyl-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-11

3-((5-bromo-6-(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-12

3-((5-bromo-6-methyl-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-13

3-((5,6-dimethoxy-1H-benzo[d] imidazol-2-yl)amino)-N-hydroxy- benzamide33-14

3-((5,6-dichloro-1H-benzo[d] imidazol-2-yl)amino)-N-hydroxy- benzamide33-15

3-((5-chloro-6-nitro-1H-benzo[d] imidazol-2-yl)amino)-N-hydroxy-benzamide 33-16

3-((6-bromo-5-fluoro-1H-benzo[d] imidazol-2-yl)amino)-N-hydroxy-benzamide 33-17

3-((5-cyano-6-(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-18

N-hydroxy-3-((7-oxo-3,6,7,8- tetrahydroimidazo[4′,5′:4,5]benzo[1,2-b][1,4]oxazin-2-yl)amino)benzamide 33-19

3-((6-chloro-5-methoxy-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-2 

3-((5-chloro-6-methyl-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-20

3-((5-chloro-1H-benzo[d]imidazol-2- yl)amino)-N-hydroxybenzamide 33-21

N-hydroxy-3-((5-nitro-1H- benzo[d]imidazol-2-yl)amino) benzamide 33-22

N-hydroxy-3-((5-(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)amino)benzamide 33-23

N-hydroxy-3-((5-(methylsulfonyl)-1H- benzo[d]imidazol-2-yl)amino)benzamide 33-24

N-hydroxy-3-((5-sulfamoyl-1H- benzo[d]imidazol-2-yl)amino) benzamide33-3 

3-((5,6-difluoro-1H-benzo[d]imidazol- 2-yl)amino)-N-hydroxybenzamide33-4 

3-((5H-[1,3]dioxolo[4′,5′:4,5]benzo [1,2-d]imidazol-6-yl)amino)-N-hydroxybenzamide 33-5 

3-((5-fluoro-6-(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-6 

3-((5-chloro-6-(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-7 

3-((5-bromo-6-(trifluoromethoxy)- 1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 33-8 

3-((7,8-dihydro-1H,6H- [1,4]dioxepino[2′,3′:4,5]benzo[1,2-d]imidazol-2-yl)amino)-N- hydroxybenzamide 33-9 

3-((6,7-dihydro-1H- [1,4]dioxino[2′,3′:4,5]benzo[1,2-d]imidazol-2-yl)amino)-N-hydroxy- benzamide 34-1 

N-hydroxy-3-(methyl(1-methyl-5- (pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 34-2 

N-hydroxy-3-(methyl(1-methyl-6- (pyridin-3-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 34-3 

N-hydroxy-3-(methyl(1-methyl-6- phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 35-1 

N-hydroxy-3-((1-methyl-5- (pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 35-2 

N-hydroxy-3-((1-methyl-6- (pyridin-3-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 35-3 

N-hydroxy-3-((1-methyl-6-phenyl-5- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide 36-1 

N-hydroxy-3-((5-phenyl-6- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide 36-10

N-hydroxy-3-((6-(3-hydroxyphenyl)- 5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide 36-11

N-hydroxy-3-((6-(2- (hydroxymethyl)phenyl)-5-(trifluoromethyl)-1H-benzo[d] imidazol-2-yl)amino)benzamide 36-12

3-((6-(6-(dimethylamino)pyridin- 3-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)- N-hydroxybenzamide 36-13

N-hydroxy-3-((6-(2-morpholino- pyrimidin-5-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 36-14

N-hydroxy-3-((6-(3-(morpholine-4- carbonyl)phenyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 36-15

3-((6-(5-fluoro-2-(hydroxymethyl) phenyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)-N- hydroxybenzamide 36-16

N-hydroxy-3-((6-(3- (methoxymethyl)phenyl)-5-(trifluoromethyl)-1H-benzo[d] imidazol-2-yl)amino)benzamide 36-17

N-hydroxy-3-((6-(1-methyl-1H- indazol-6-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 36-18

3-(2-((3-(hydroxycarbamoyl) phenyl)amino)-5-(trifluoromethyl)-1H-benzo[d]imidazol-6-yl)-N,N- dimethylbenzamide 36-19

N-hydroxy-3-((6-(4-morpholino- phenyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 36-2 

N-hydroxy-3-((6-(pyridin-3-yl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide 36-20

3-((6-(furan-3-yl)-5-(trifluoro- methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 36-3 

N-hydroxy-3-((5-(pyridin-4-yl)-6- (trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide 36-4 

3-((6-(3-ethoxyphenyl)-5-(trifluoro- methyl)-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide 36-5 

N-hydroxy-3-((6-(4-(methylthio) phenyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 36-6 

4-(2-((3-(hydroxycarbamoyl) phenyl)amino)-5-(trifluoro-methyl)-1H-benzo[d]imidazol-6- yl)-N,N-dimethylbenzamide 36-7 

N-hydroxy-3-((6-(4- (methoxymethyl)phenyl)-5-(trifluoromethyl)-1H-benzo[d] imidazol-2-yl)amino)benzamide 36-8 

N-hydroxy-3-((6-(quinolin-6-yl)- 5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide 36-9 

3-((6-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl) amino)-N-hydroxybenzamide 37-1 

N-((benzylcarbamoyl)oxy)-3-((5- phenyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino) benzamide 37-2 

N-((benzylcarbamoyl)oxy)-3-((5- cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)(methyl) amino)benzamide or a pharmaceuticallyacceptable salt thereof.

Methods of Synthesizing the Disclosed Compounds

The compounds of the present invention may be made by a variety ofmethods, including standard chemistry. Suitable synthetic routes aredepicted in the schemes given below.

The compounds of Formula I may be prepared by methods known in the artof organic synthesis as set forth in part by the following syntheticschemes and examples. In the schemes described below, it is wellunderstood that protecting groups for sensitive or reactive groups areemployed where necessary in accordance with general principles orchemistry. Protecting groups are manipulated according to standardmethods of organic synthesis (T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999). These groups are removed at a convenient stage of the compoundsynthesis using methods that are readily apparent to those skilled inthe art. The selection processes, as well as the reaction conditions andorder of their execution, shall be consistent with the preparation ofcompounds of Formula I.

Those skilled in the art will recognize if a stereocenter exists in thecompounds of Formula I. Accordingly, the present invention includes bothpossible stereoisomers (unless specified in the synthesis) and includesnot only racemic compounds but the individual enantiomers and/ordiastereomers as well. When a compound is desired as a single enantiomeror diastereomer, it may be obtained by stereospecific synthesis or byresolution of the final product or any convenient intermediate.Resolution of the final product, an intermediate, or a starting materialmay be affected by any suitable method known in the art. See, forexample, “Stereochemistry of Organic Compounds” by E. L. Eliel, S. H.Wilen, and L. N. Mander (Wiley-lnterscience, 1994).

Preparation of Compounds

The compounds described herein may be made from commercially availablestarting materials or synthesized using known organic, inorganic, and/orenzymatic processes.

The compounds of the present invention can be prepared in a number ofways well known to those skilled in the art of organic synthesis. By wayof example, compounds of the Formula I can be synthesized using themethods described below, together with synthetic methods known in theart of synthetic organic chemistry, or variations thereon as appreciatedby those skilled in the art. These methods include but are not limitedto those methods described below.

wherein X₁, X₂, X₃, X₄, and Z are described herein.

A general way of preparing the compounds of the present invention usingan amine 1 is outlined in General Scheme I-1. Condensation andcyclization of amine 1 with compound 2 will provide the carboxylate 3. Afinal condensation of compound 3 with a hydroxyamine will generallyprovide the compounds of formula I.

wherein X₁, X₂, X₃, X₄, and Z are described herein.

Another general way of preparing the compounds of the present inventionis outlined in General Scheme II-1. Cross-coupling of amine 1 withcompound 6 under standard conditions using metal-catalyzed coupling willprovide the carboxylate 7. A final condensation of compound 7 with ahydroxyamine will generally provide the compounds of formula I.

wherein X₁, X₂, X₃, X₄, and Z are described herein.

Another general way of preparing the compounds of the present inventionis outlined in General Scheme III-1. Cross-coupling of compound 1 withamine 9 under standard conditions using metal-catalyzed coupling willprovide the carboxylate 10. A final condensation of compound 10 with ahydroxyamine will generally provide the compounds of formula I.

Spirocycles

One aspect of the invention relates to compounds of Formula II:

and pharmaceutically acceptable salts thereof, wherein:

X¹, X², X³, X⁴, X⁵, and X⁶ are each independently, at each occurrence,—CR¹R²—, —NR³—, —O—, —C(O)—, —S(O)₂—, —S(O)—, or —S—;

Y¹, Y², and Y³ are each independently N or CR¹;

L is a bond, —(CR¹R²)_(p)—, —C(O)NR³—, —S(O)₂—, —S(O)₂NR³—, —S(O)—,—S(O)NR³—, —C(O)(CR¹R²)_(p)O—, or —C(O)(CR¹R²)_(p)—;

R is independently —H, —C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl,—C₂-C₆alkynyl, —C₃-C₈cycloalkyl, —C₅-C₁₂spirocycle, heterocyclyl,spiroheterocyclyl, aryl, or heteroaryl containing 1-5 heteroatomsselected from the group consisting of N, S, P, or O, wherein each alkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkyl, spirocycle, heterocyclyl,spiroheterocyclyl, aryl, or heteroaryl is optionally substituted withone or more —OH, halogen, oxo, —NO₂, —CN, —R¹, —R², —SR³, —OR³, —NHR³,—NR³R⁴, —S(O)₂NR³R⁴, —S(O)₂R¹, —C(O)R¹, —C(O)OR¹, —NR³S(O)₂R¹, —S(O)R¹,—S(O)NR³R⁴, —NR³S(O)R¹, heterocycle, aryl, or heteroaryl;

R¹ and R² are independently, at each occurrence, —H, —R³, —R⁴,—C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,—C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5heteroatoms selected from the group consisting of N, S, P and O, —OH,halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,—S(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)₂R⁵, —S(O)₂(C₁-C₆alkyl),—(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,—N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, or —(CHR⁵)_(p)NR³R⁴, wherein each alkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with one or more —OH, halogen,—NO₂, oxo, —CN, —R⁵, —OR³, —NHR³, —NR³R⁴, —S(O)₂N(R³)₂—, —S(O)₂R⁵,—C(O)R⁵, —C(O)OR⁵, —NR³S(O)₂R⁵, —S(O)R⁵, —S(O)NR³R⁴, —NR³S(O)R⁵,heterocycle, aryl, or heteroaryl;

or R¹ and R² can combine with the carbon atom to which they are bothattached to form a spirocycle, spiroheterocycle, or spirocycloalkenyl;

or R¹ and R², when on adjacent atoms, can combine to form a cycloalkyl,a heterocycle, aryl, heteroaryl containing 1-5 heteroatoms selected fromthe group consisting of N, S, P and O, or a cycloalkenyl;

or R¹ and R², when on non-adjacent atoms, can combine to form anoptionally bridging cycloalkyl, an optionally bridging heterocycle, oran optionally bridging cycloalkenyl;

R³ and R⁴ are independently, at each occurrence, —H, —C₁-C₆alkyl,—C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl,heterocyclyl, aryl, heteroaryl containing 1-5 heteroatoms selected fromN, S, P, and O, —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂(C₁-C₆alkyl),—(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, or—(CHR⁵)_(p)N(C₁-C₆alkyl)₂, wherein each alkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionallysubstituted with one or more substituents selected from —OH, halogen,—NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl, —N(C₁-C₆alkly)₂,—S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl,—C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵,—S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, orheteroaryl;

R⁵ is independently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆alkenyl,—C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,heteroaryl containing 1-5 heteroatoms selected from N, S, P and O, —OH,halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,—S(O)₂NH(C₁-C₆alkyl), —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂C₁-C₆alkyl,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)SO₂C₁-C₆alkyl,—S(O)(C₁-C₆alkyl), —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)(C₁-C₆alkyl)or —(CH₂)_(p)N(C₁-C₆alkyl)₂;

p is 0, 1, 2, 3, 4, 5, or 6;

n is 0, 1, 2, 3, or 4;

m is 0, 1, or 2;

q is 1 or 2;

r is 1 or 2;

wherein the sum q+r≤3 and

wherein the sum m+n≤4.

In one or more embodiments, n is 0 and m is 1. In one or moreembodiments, n is 1 and m is 1. In one or more embodiments, q is 1 and ris 1. In one or more embodiments, q is 2 and r is 1. In one or moreembodiments, q is 1 and r is 2. In one or more embodiments, q is 1, r is1, m is 0 and n is 1. In one or more embodiments, q is 1, r is 1, m is 1and n is 1. In one or more embodiments, q is 1, r is 1, m is 2 and nis 1. In one or more embodiments, q is 1, r is 1, m is 1 and n is 2. Inone or more embodiments, X⁵ is C(O). In one or more embodiments, X⁴ isC(O).

In some embodiments, X¹ is —CH₂—. In some embodiments, X¹ is —CH₂—, andX² is —CH₂—. In some embodiments, X¹ is —CH₂—, X² is —CH₂—, and X³ is—CH₂—. In some embodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, andX⁴—CH₂—. In some embodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴is —CH₂—, and X⁵ is —CH₂—. In some embodiments, X¹ is —CH₂—, X² is—CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is —CH₂—,X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0,and n is 0. In some embodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—,X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and q is 1. Insome embodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —O—. In some embodiments, X¹ is —O—, and X²is —CH₂—. In some embodiments, X¹ is —O—, X² is —CH₂—, and X³ is —CH₂—.In some embodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, and X⁴—CH₂—.In some embodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,and X⁵ is —CH₂—. In some embodiments, X1 is —O—, X² is —CH₂—, X³ is—CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is —O—, X²is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0,and n is 0. In some embodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and q is 1. In someembodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. In someembodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. In someembodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. In someembodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —O—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —O—, and X² is —O—. In some embodiments, X¹is —O—, X² is —O—, and X³ is —CH₂—. In some embodiments, X¹ is —O—, X²is —O—, X³ is —CH₂—, and X⁴—CH₂—. In some embodiments, X¹ is —O—, X² is—O—, X³ is —CH₂—, X⁴ is —CH₂—, and X⁵ is —CH₂—. In some embodiments, X1is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, and X⁶ is—CH₂—.

In some embodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is —O—, X² is—O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, and nis 0. In some embodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and q is 1. In someembodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. In someembodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is. In someembodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is. In someembodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. In someembodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —O—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —CH₂—, and X² is —O—. In some embodiments, X¹is —CH₂—, X² is —O—, and X³ is —CH₂—. In some embodiments, X¹ is —CH₂—,X² is —O—, X³ is —CH₂—, and X⁴—CH₂—. In some embodiments, X¹ is —CH₂—,X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, and X⁵ is —CH₂—. In someembodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is —CH₂—,X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0,and n is 0. In some embodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and q is 1. In someembodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —O—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —NR³—. In some embodiments, X¹ is —NR³—, andX² is —CH₂—. In some embodiments, X¹ is —NR³—, X² is —CH₂—, and X³ is—CH₂—. In some embodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, andX⁴—CH₂—. In some embodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴is —CH₂—, and X⁵ is —CH₂—. In some embodiments, X¹ is —NR³—, X² is—CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is —NR³—,X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0,and n is 0. In some embodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—,X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and q is 1. Insome embodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. In someembodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. In someembodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. In someembodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —NR³—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —S(O)—. In some embodiments, X¹ is —S(O)—,and X² is —CH₂—. In some embodiments, X¹ is —S(O)—, X² is —CH₂—, and X³is —CH₂—. In some embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—,and X⁴—CH₂—. In some embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is—CH₂—, X⁴ is —CH₂—, and X⁵ is —CH₂—. In some embodiments, X¹ is —S(O)—,X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is—S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—,m is 0, and n is 0. In some embodiments, X¹ is —S(O)—, X² is —CH₂—, X³is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and qis 1. In some embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. Insome embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. Insome embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. Insome embodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —S(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —S(O)₂—. In some embodiments, X¹ is —S(O)₂—,and X² is —CH₂—. In some embodiments, X¹ is —S(O)₂—, X² is —CH₂—, and X³is —CH₂—. In some embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—,and X⁴—CH₂— In some embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is—CH₂—, X⁴ is —CH₂—, and X⁵ is —CH₂—. In some embodiments, X^(1′) is—S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, and X⁶ is—CH₂—.

In some embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is—S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is—CH₂—, m is 0, and n is 0. In some embodiments, X¹ is —S(O)₂—, X² is—CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is0, and q is 1. In some embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is—CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1,and r is 1.

In some embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. Insome embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. Insome embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. Insome embodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —S(O)₂—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —C(O)—. In some embodiments, X¹ is —C(O)—,and X² is —CH₂—. In some embodiments, X¹ is —C(O)—, X² is —CH₂—, and X³is —CH₂—. In some embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—,and X⁴—CH₂—. In some embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is—CH₂—, X⁴ is —CH₂—, and X⁵ is —CH₂—. In some embodiments, X¹ is —C(O)—,X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is—C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—,m is 0, and n is 0. In some embodiments, X¹ is —C(O)—, X² is —CH₂—, X³is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and qis 1. In some embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. Insome embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. Insome embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. Insome embodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —C(O)—, X² is —CH₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —CH₂—, and X² is —NR³—. In some embodiments,X¹ is —CH₂—, X² is —NR³—, and X³ is —CH₂—. In some embodiments, X¹ is—CH₂—, X² is —NR³—, X³ is —CH₂—, and X⁴—CH₂—. In some embodiments, X¹ is—CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, and X⁵ is —CH₂—. In someembodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is —CH₂—,X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0,and n is 0. In some embodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—,X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and q is 1. Insome embodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —NR³—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —CH₂—, and X² is —S(O)—. In some embodiments,X¹ is —CH₂—, X² is —S(O)—, and X³ is —CH₂—. In some embodiments, X¹ is—CH₂—, X² is —S(O)—, X³ is —CH₂—, and X⁴—CH₂—. In some embodiments, X¹is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—, and X⁵ is —CH₂—. Insome embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is—CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—,m is 0, and n is 0. In some embodiments, X¹ is —CH₂—, X² is —S(O)—, X³is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and qis 1. In some embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. Insome embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. Insome embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. Insome embodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —S(O)—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —CH₂—, and X² is —S(O)₂—. In someembodiments, X¹ is —CH₂—, X² is —S(O)₂—, and X³ is —CH₂—. In someembodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, and X⁴—CH₂—. Insome embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—,and X⁵ is —CH₂—. In some embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is—CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is—CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is—CH₂—, m is 0, and n is 0. In some embodiments, X¹ is —CH₂—, X² is—S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, nis 0, and q is 1. In some embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is—CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1,and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. Insome embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. Insome embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. Insome embodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —S(O)₂—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, X¹ is —CH₂—, and X² is —C(O)—. In some embodiments,X¹ is —CH₂—, X² is —C(O)—, and X³ is —CH₂—. In some embodiments, X¹ is—CH₂—, X² is —C(O)—, X³ is —CH₂—, and X⁴—CH₂—. In some embodiments, X¹is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—, and X⁵ is —CH₂—. Insome embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, and X⁶ is —CH₂—.

In some embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, and m is 0. In some embodiments, X¹ is—CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—,m is 0, and n is 0. In some embodiments, X¹ is —CH₂—, X² is —C(O)—, X³is —CH₂—, X⁴ is —CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, and qis 1. In some embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 0, n is 0, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 1. Insome embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 1.

In some embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 1, and r is 2. Insome embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 1, and r is 2. In someembodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 1, and r is 2.

In some embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is—CH₂—, X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 0, q is 2, and r is 1. Insome embodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—,X⁵ is —CH₂—, X⁶ is —CH₂—, m is 1, n is 1, q is 2, and r is 1. In someembodiments, X¹ is —CH₂—, X² is —C(O)—, X³ is —CH₂—, X⁴ is —CH₂—, X⁵ is—CH₂—, X⁶ is —CH₂—, m is 0, n is 1, q is 2, and r is 1.

In some embodiments, Y¹ is —CH—. In some embodiments, Y² is —CH—. Insome embodiments, Y³ is —CH—. In some embodiments, Y¹ is —N—. In someembodiments, Y² is —N—. In some embodiments, Y³ is —N—. In someembodiments, Y¹ is —CH—, Y² is —CH—, and, and Y³ is —CH—. In someembodiments, Y¹ is —N—, Y² is —CH—, and, and Y³ is —N—. In someembodiments, Y¹ is —N—, Y² is —N—, and, and Y³ is —CH—. In someembodiments, Y¹ is —CH—, Y² is —N—, and, and Y³ is —CH—. In someembodiments, Y¹ is —CH—, Y² is —N—, and, and Y³ is —N—.

In some embodiments, X⁴ is —C(O)— and X¹, X², X³, X⁵, X⁶ are —CH₂—. Insome embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0 andn is 0. In some embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—,m is 1 and n is 0. In some embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶are —CH₂—, m is 0 and n is 1. In some embodiments, X⁴ is —C(O)—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0,n is 0, q is 1 and r is 1. In some embodiments, X⁴ is —C(O)—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1 and r is 1. In someembodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is 1,q is 1 and r is 1. In some embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶are —CH₂—, m is 1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0,n is 0, q is 2 and r is 1. In some embodiments, X⁴ is —C(O)—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2 and r is 1. In someembodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is 1,q is 2 and r is 1. In some embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶are —CH₂—, m is 1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0,n is 0, q is 1 and r is 2. In some embodiments, X⁴ is —C(O)—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1 and r is 2. In someembodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is 1,q is 1 and r is 2. In some embodiments, X⁴ is —C(O)—, X¹, X², X³, X⁵, X⁶are —CH₂—, m is 1, n is 1, q is 1 and r is 2.

In some embodiments, X⁴ is —S(O)— and X¹, X², X³, X⁵, X⁶ are —CH₂—. Insome embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0 andn is 0. In some embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—,m is 1 and n is 0 In some embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶are —CH₂—, m is 0 and n is 1. In some embodiments, X⁴ is —S(O)—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0,n is 0, q is 1 and r is 1. In some embodiments, X⁴ is —S(O)—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1 and r is 1. In someembodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is 1,q is 1 and r is 1. In some embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶are —CH₂—, m is 1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0,n is 0, q is 2 and r is 1. In some embodiments, X⁴ is —S(O)—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2 and r is 1. In someembodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is 1,q is 2 and r is 1. In some embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶are —CH₂—, m is 1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0,n is 0, q is 1 and r is 2. In some embodiments, X⁴ is —S(O)—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1 and r is 2. In someembodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is 1,q is 1 and r is 2. In some embodiments, X⁴ is —S(O)—, X¹, X², X³, X⁵, X⁶are —CH₂—, m is 1, n is 1, q is 1 and r is 2.

In some embodiments, X⁴ is —S(O)₂— and X¹, X², X³, X⁵, X⁶ are —CH₂—. Insome embodiments, X⁴ is —S(O)₂—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0and n is 0. In some embodiments, X⁴ is —S(O)₂—, X¹, X², X³, X⁵, X⁶ are—CH₂—, m is 1 and n is 0. In some embodiments, X⁴ is —S(O)₂—, X¹, X²,X³, X⁵, X⁶ are —CH₂—, m is 0 and n is 1. In some embodiments, X⁴ is—S(O)₂—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —S(O)₂—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is0, n is 0, q is 1 and r is 1. In some embodiments, X⁴ is —S(O)₂—, X¹,X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1 and r is 1. In someembodiments, X⁴ is —S(O)₂—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is1, q is 1 and r is 1. In some embodiments, X⁴ is —S(O)₂—, X¹, X², X³,X⁵, X⁶ are —CH₂—, m is 1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —S(O)₂—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is0, n is 0, q is 2 and r is 1. In some embodiments, X⁴ is —S(O)₂—, X¹,X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2 and r is 1. In someembodiments, X⁴ is —S(O)₂—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is1, q is 2 and r is 1. In some embodiments, X⁴ is —S(O)₂—, X¹, X², X³,X⁵, X⁶ are —CH₂—, m is 1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —S(O)₂—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is0, n is 0, q is 1 and r is 2. In some embodiments, X⁴ is —S(O)₂—, X¹,X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1 and r is 2. In someembodiments, X⁴ is —S(O)₂—, X¹, X², X³, X⁵, X⁶ are —CH₂—, m is 0, n is1, q is 1 and r is 2. In some embodiments, X⁴ is —S(O)₂—, X¹, X², X³,X⁵, X⁶ are —CH₂—, m is 1, n is 1, q is 1 and r is 2.

In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—. In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶are —CH₂—, m is 0 and n is 0. In some embodiments, X⁴ is —C(O)—, X¹ is—O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 0. In someembodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is0 and n is 1. In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 1. In some embodiments, X⁴ is—C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 1. In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 1. In someembodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 2 and r is 1. In some embodiments, X⁴ is—C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2and r is 1. In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 2 and r is 1. In someembodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 2. In some embodiments, X⁴ is—C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 2. In some embodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 2. In someembodiments, X⁴ is —C(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 1 and r is 2.

In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—. In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶are —CH₂—, m is 0 and n is 0. In some embodiments, X⁴ is —S(O)—, X¹ is—O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 0. In someembodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is0 and n is 1. In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 1. In some embodiments, X⁴ is—S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 1. In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 1. In someembodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 2 and r is 1. In some embodiments, X⁴ is—S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2and r is 1. In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 2 and r is 1. In someembodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 2. In some embodiments, X⁴ is—S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 2. In some embodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 2. In someembodiments, X⁴ is —S(O)—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 1 and r is 2.

In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—. In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶are —CH₂—, m is 0 and n is 0. In some embodiments, X⁴ is —S(O)₂—, X¹ is—O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 0. In someembodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, mis 0 and n is 1. In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X²,X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 1. In some embodiments, X⁴ is—S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 1. In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 1. In someembodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, mis 1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 2 and r is 1. In some embodiments, X⁴ is—S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2and r is 1. In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 2 and r is 1. In someembodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, mis 1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 2. In some embodiments, X⁴ is—S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 2. In some embodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 2. In someembodiments, X⁴ is —S(O)₂—, X¹ is —O—, and X², X³, X⁵, X⁶ are —CH₂—, mis 1, n is 1, q is 1 and r is 2.

In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—. In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶are —CH₂—, m is 0 and n is 0. In some embodiments, X⁴ is —C(O)—, X² is—O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 0. In someembodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is0 and n is 1. In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 1. In some embodiments, X⁴ is—C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 1. In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 1. In someembodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 2 and r is 1. In some embodiments, X⁴ is—C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2and r is 1. In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 2 and r is 1. In someembodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 2. In some embodiments, X⁴ is—C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 2. In some embodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 2. In someembodiments, X⁴ is —C(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 1 and r is 2.

In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—. In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶are —CH₂—, m is 0 and n is 0. In some embodiments, X⁴ is —S(O)—, X² is—O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 0. In someembodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is0 and n is 1. In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 1. In some embodiments, X⁴ is—S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 1. In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 1. In someembodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 2 and r is 1. In some embodiments, X⁴ is—S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2and r is 1. In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 2 and r is 1. In someembodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 2. In some embodiments, X⁴ is—S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 2. In some embodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 2. In someembodiments, X⁴ is —S(O)—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is1, n is 1, q is 1 and r is 2.

In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—. In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶are —CH₂—, m is 0 and n is 0. In some embodiments, X⁴ is —S(O)₂—, X² is—O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 0. In someembodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, mis 0 and n is 1. In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹,X³, X⁵, X⁶ are —CH₂—, m is 1 and n is 1.

In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 1. In some embodiments, X⁴ is—S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 1. In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 1. In someembodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, mis 1, n is 1, q is 1 and r is 1.

In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 2 and r is 1. In some embodiments, X⁴ is—S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 2and r is 1. In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 2 and r is 1. In someembodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, mis 1, n is 1, q is 2 and r is 1.

In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are—CH₂—, m is 0, n is 0, q is 1 and r is 2. In some embodiments, X⁴ is—S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, m is 1, n is 0, q is 1and r is 2. In some embodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³,X⁵, X⁶ are —CH₂—, m is 0, n is 1, q is 1 and r is 2. In someembodiments, X⁴ is —S(O)₂—, X² is —O—, and X¹, X³, X⁵, X⁶ are —CH₂—, mis 1, n is 1, q is 1 and r is 2.

In one or more embodiments, the compound is of the Formula II-A:

In one or more embodiments, the compound is of the Formula II-B:

In one or more embodiments, the compound is of the Formula II-C:

In one or more embodiments, the compound is of the Formula II-D:

In one or more embodiments, the compound is of the Formula II-E:

For each of the embodiments described above for compounds of Formula II,II-A, II-B, II-C, II-D, or II-E, L is selected from a bond, —CH₂—,—(CH₂)₂—, —(CH₂)₃—, —C(O)—, —S(O)₂—, —C(O)NR³—, or —C(O)CH₂—. For eachof the embodiments described above for compounds of Formula II, II-A,II-B, II-C, II-D, or II-E, L is selected from a bond, —CH₂—, or —C(O)—.For each of the embodiments for compounds of Formula II, II-A, II-B,II-C, II-D, or II-E, L is selected from a bond. For each of theembodiments for compounds of Formula II, II-A, II-B, II-C, II-D, orII-E, L is selected from —CH₂—. For each of the embodiments forcompounds of Formula II, II-A, II-B, II-C, II-D, or II-E, L is selectedfrom —C(O)—.

For each of the embodiments described above for compounds of Formula II,II-A, II-B, II-C, II-D, or II-E, R is hydrogen or an optionallysubstituted group selected from C₁-C₆alkyl, aryl, C₃-C₈ cycloalkyl,heterocyclyl, or heteroaryl containing 1-5 heteroatoms selected from thegroup consisting of N, S, P, or O. For each of the embodiments describedabove for compounds for Formula II, II-A, II-B, II-C, II-D, or II-E, Ris hydrogen or an optionally substituted group selected from phenyl,C₁-C₆alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyridyl,or morpholinyl. For each of the embodiments described above forcompounds of Formula II, II-A, II-B, II-C, II-D, or II-E, R is phenyloptionally substituted with one or more independent occurrences ofhalogen, CF₃, —SO₂CH₃, phenyl, C₁-C₃ alkyl, C₁-C₃alkoxy, pyridyl, —OCF₃or —OCH₂phenyl.

In one or more embodiments, the compound is of the Formula II-A-i:

In one or more embodiments, the compound is of the Formula II-B-i:

In one or more embodiments, the compound is of the Formula II-C-i:

In one or more embodiments, the compound is of the Formula II-C-ii:

In one or more embodiments, the compound is of the Formula II-D-i:

In one or more embodiments, the compound is of the Formula II-E-i:

For each of the embodiments described above for compounds of Formula II,II-A-i, II-B-i, II-C-i, II-C-ii, II-D-i, or II-E-i, L is selected from abond, —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —C(O)—, —S(O)₂—, —C(O)NR³—, or—C(O)CH₂—. For each of the embodiments described above for compounds ofFormula II, II-A-i, II-B-i, II-C-i, II-C-ii, II-D-i, or II-E-i, L isselected from a bond, —CH₂—, or —C(O)—. For each of the embodiments forcompounds of Formula II, II-A-i, II-B-i, II-C-i, II-C-ii, II-D-i, orII-E-i, L is selected from a bond. For each of the embodiments forcompounds of Formula II, II-A-i, II-B-i, II-C-i, II-C-ii, II-D-i, orII-E-i, L is selected from —CH₂—. For each of the embodiments forcompounds of Formula II, II-A-i, II-B-i, II-C-i, II-C-ii, II-D-i, orII-E-i, L is selected from —C(O)—.

For each of the embodiments described above for compounds of Formula II,II-A-i, II-B-i, II-C-i, II-C-ii, II-D-i, or II-E-i, R is hydrogen or anoptionally substituted group selected from C₁-C₆alkyl, aryl, C₃-C₈cycloalkyl, heterocyclyl, or heteroaryl containing 1-5 heteroatomsselected from the group consisting of N, S, P, or O. For each of theembodiments described above for compounds for Formula II, II-A-i,II-B-i, II-C-i, II-C-ii, II-D-i, or II-E-i, R is hydrogen or anoptionally substituted group selected from phenyl, C₁-C₆alkyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyridyl, ormorpholinyl. For each of the embodiments described above for compoundsof Formula II, II-A-i, II-B-i, II-C-i, II-C-ii, II-D-i, or II-E-i, R isphenyl optionally substituted with one or more independent occurrencesof halogen, CF₃, —SO₂CH₃, phenyl, C₁-C₃ alkyl, C₁-C₃alkoxy, pyridyl,—OCF₃ or —OCH₂phenyl.

In one or more embodiments, a compound of Formula II can be selectedfrom:

-   1′-(4-chlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-1);-   N-hydroxy-1′-methyl-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-2);-   (R)-1′-(4-chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-3);-   (S)-1′-(4-chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (HDTK054);-   1′-(3-fluoro-4-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-5);-   N-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-6);-   1′-(4-fluorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-7);-   N-hydroxy-1′-(4-(methyl    sulfonyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-8);-   1′-cyclopropyl-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-9);-   1′-(cyclobutylmethyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-10);-   N-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-11);-   N-hydroxy-2′-oxo-1′-(3-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-12);-   N-hydroxy-2′-oxo-1′-(2-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-13);-   1′-benzyl-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-14);-   N-hydroxy-2′-oxo-1′-(3-phenylpropyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-15);-   1′-([1,1′-biphenyl]-4-ylmethyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-16);-   N-hydroxy-1′-(3-methylbenzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-17);-   1′-([1,1′-biphenyl]-3-ylmethyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-18);-   N-hydroxy-1′-(4-methoxybenzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-19);-   N-hydroxy-1′-(3-methoxybenzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-20);-   1′-(2,6-dichlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-21);-   1′-(2,3-dichlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-22);-   N-hydroxy-1′-(2-methylbenzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-23);-   N-hydroxy-2′-oxo-1′-(pyridin-3-ylmethyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-24);-   1′-(2-chlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-25);-   1′-(3-fluorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-26);-   N-hydroxy-2′-oxo-1′-(4-(trifluoromethoxy)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-27);-   1′-(3,5-difluorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-28);-   N-hydroxy-2′-oxo-1′-(2-(trifluoromethyl)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-29);-   1′-(3-chlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-30);-   N-hydroxy-2′-oxo-1′-(3-(trifluoromethyl)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-31);-   N-hydroxy-2′-oxo-1′-(2-(trifluoromethoxy)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-32);-   1′-(3-bromobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-33);-   N-hydroxy-2′-oxo-1′-((2-phenylthiazol-4-yl)methyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-34);-   1′-(2,4-difluorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-35);-   N-hydroxy-1′-(2-methoxybenzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-36);-   1′-([1,1′-biphenyl]-2-ylmethyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-37);-   1′-(3,4-dichlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-38);-   1′-(3,4-dimethylbenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-39);-   1′-(3,5-dimethoxybenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-40);-   1′-(4-(benzyloxy)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-41);-   N-hydroxy-2′-oxo-1′-(3-(trifluoromethoxy)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-42);-   1′-(2-fluoro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-43);-   1′-(cyclohexylmethyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-44);-   1′-(2,5-dichlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-45);-   N-hydroxy-2′-oxo-1′-((3-phenylisoxazol-5-yl)methyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-46);-   1′-((6-(1H-pyrazol-1-yl)pyridin-3-yl)methyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-47);-   N-hydroxy-2′-oxo-1′-((6-(trifluoromethyl)pyridin-3-yl)methyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-48);-   N-hydroxy-2′-oxo-1′-((5-(tetrahydro-2H-pyran-4-yl)-1,2,4-oxadiazol-3-yl)methyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-49);-   1′-((3,5-dimethylisoxazol-4-yl)methyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-50);-   N-hydroxy-2′-oxo-1′-((6-(trifluoromethyl)pyridin-2-yl)methyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-51);-   N-hydroxy-1′-(2-morpholinoethyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-52);-   N-hydroxy-2′-oxo-1′-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-53);-   N-hydroxy-1′-(4-(methylsulfonyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-54);-   1′-(cyclopropylmethyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-55);-   N-hydroxy-1′-(4-(trifluoromethyl)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-56);-   1′-cyclopropyl-N-hydroxy-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-57);-   N-hydroxy-1′-(4-(trifluoromethyl)benzoyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-58);-   1′-acetyl-N-hydroxy-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide    (II-59);-   1′-acetyl-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide    (II-60);-   1′-(4-chlorobenzoyl)-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide    (II-61);-   1′-(4-chlorobenzyl)-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide    (II-62);-   1′-cyclopropyl-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide    (II-63);-   1′-benzyl-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide    (II-64);-   1′-benzyl-N-hydroxy-1-oxo-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide    (II-65);-   1′-benzyl-N,    1-dihydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide    (II-66);-   1′-(4-chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,    3′-pyrrolidine]-8-carboxamide (II-67);-   1′-(3-fluoro-4-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,    3′-pyrrolidine]-8-carboxamide (II-68);-   1′-(4-chlorobenzyl)-N-hydroxy-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide    (II-69);-   N-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)benzyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide    (II-70);-   N-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide    (II-71);-   N-hydroxy-2′-oxo-1′-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide    (II-72);-   N-hydroxy-2′-oxo-1′-(2-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide    (II-73);-   N8-hydroxy-N1′,N1′-dimethylspiro[chromane-2,4′-piperidine]-1′,8-dicarboxamide    (II-74);-   1′-(4-fluorobenzoyl)-N-hydroxyspiro[chromane-2,4′-piperidine]-8-carboxamide    (II-75);-   1′-(cyclohexylsulfonyl)-N-hydroxyspiro[chromane-2,4′-piperidine]-8-carboxamide    (II-76);-   N-hydroxy-1′-(4-methyltetrahydro-2H-pyran-4-carbonyl)spiro[chromane-2,4′-piperidine]-8-carboxamide    (II-77);-   N-hydroxy-1′-(1-(3-(trifluoromethyl)phenyl)cyclopropane-1-carbonyl)spiro[chromane-2,4′-piperidine]-8-carboxamide    (II-78);-   N-hydroxy-1′-(2-phenylacetyl)spiro[chromane-2,4′-piperidine]-8-carboxamide    (II-79);-   1′-(4-fluorobenzyl)-N-hydroxyspiro[chromane-2,4′-piperidine]-8-carboxamide    (II-80);-   1′-cyclohexyl-N-hydroxyspiro[chromane-2,4′-piperidine]-8-carboxamide    (II-81);-   N-hydroxy-1′-(5-(trifluoromethyl)pyridin-2-yl)spiro[chromane-2,4′-piperidine]-8-carboxamide    (II-82);-   1′-(3-fluoro-4-methylphenyl)-N-hydroxyspiro[chromane-2,4′-piperidine]-8-carboxamide    (II-83);-   1′-(4-chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxamide    (II-84);-   N-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)benzyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxamide    (II-85);-   1′-(3-fluoro-4-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxamide    (II-86);-   1′-(4-chlorobenzyl)-N-hydroxy-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxamide    (II-87);-   N-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxamide    (II-88);-   N-hydroxy-2′-oxo-1′-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxamide    (II-89);-   N-hydroxy-2′-oxo-1′-(2-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxamide    (II-90);-   1′-acetyl-N-hydroxyspiro[chromane-2,4′-piperidine]-5-carboxamide    (II-91);-   N-hydroxy-1′-phenylspiro[chromane-2,4′-piperidine]-5-carboxamide    (II-92) or-   1′-(4-fluorophenyl)-N-hydroxyspiro[chromane-2,4′-piperidine]-5-carboxamide    (II-93),

or a pharmaceutically acceptable salt thereof.

Methods of Synthesizing the Disclosed Compounds

The compounds of the present invention may be made by a variety ofmethods, including standard chemistry. Suitable synthetic routes aredepicted in the schemes given below.

The compounds of Formula II may be prepared by methods known in the artof organic synthesis as set forth in part by the following syntheticschemes and examples. In the schemes described below, it is wellunderstood that protecting groups for sensitive or reactive groups areemployed where necessary in accordance with general principles orchemistry. Protecting groups are manipulated according to standardmethods of organic synthesis (T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999). These groups are removed at a convenient stage of the compoundsynthesis using methods that are readily apparent to those skilled inthe art. The selection processes, as well as the reaction conditions andorder of their execution, shall be consistent with the preparation ofcompounds of Formula II.

Those skilled in the art will recognize if a stereocenter exists in thecompounds of Formula II. Accordingly, the present invention includesboth possible stereoisomers (unless specified in the synthesis) andincludes not only racemic compounds but the individual enantiomersand/or diastereomers as well. When a compound is desired as a singleenantiomer or diastereomer, it may be obtained by stereospecificsynthesis or by resolution of the final product or any convenientintermediate. Resolution of the final product, an intermediate, or astarting material may be affected by any suitable method known in theart. See, for example, “Stereochemistry of Organic Compounds” by E. L.Eliel, S. H. Wilen, and L. N. Mander (Wiley-lnterscience, 1994).

Preparation of Compounds

The compounds described herein may be made from commercially availablestarting materials or synthesized using known organic, inorganic, and/orenzymatic processes.

The compounds of the present invention can be prepared in a number ofways well known to those skilled in the art of organic synthesis. By wayof example, compounds of the Formula II can be synthesized using themethods described below, together with synthetic methods known in theart of synthetic organic chemistry, or variations thereon as appreciatedby those skilled in the art. These methods include but are not limitedto those methods described below.

wherein L and R are defined as in Formula (II).

A general method of preparing target molecules of Formula (II-A) byusing Intermediates 1a-2, 1b-2, 1c-2, 1d-2, 1e-2, 1f-2, and 1g-2 isoutlined in Scheme 1-2. Carbonylation of the starting material (1a-2),for instance in the presence of a metal catalyst, e.g.,[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)(Pd(dppf)Cl₂), carbon monoxide, and a base, e.g., triethylamine (Et₃N),provides ester 1b-2. Further carbonylation of 1b-2, for instance using abase, e.g., sodium hydride (NaH), in the presence of dimethyl carbonateaffords Intermediate 1c-2, which can be alkylated by treatment with ahalo-nitrile in the presence of a base to provide Intermediates 1d-2.Reduction, for example, with hydrogen gas in the presence of platinum(IV) oxide (PtO₂), acetic acid, and methanol (MeOH), can provide alkylamine 1e-2. Spiro-lactams 1f-2 can be obtained, for example, bytreatment of 1e-2 with ammonia (NH₃) in MeOH. Addition of the R-L moietycan be achieved via standard methods of, for instance, alkylation,arylation, acylation, urea formation, or sulfonation. For example,alkylation of 1f-2 with an alkyl halide in the presence of a base, e.g.,NaH, can provide compounds of Intermediates 1g-2. Alternatively,arylation of 1f-2 with an aryl halide in the presence of a metalcatalyst, e.g., copper (I) iodide (CuI), a diamine ligand, and a base,can also provide compounds of Intermediates 1g-2. Treatment of 1g-2 withhydroxylamine and a base, e.g., sodium hydroxide (NaOH), can providecompounds of Formula (II-A).

wherein L and R are defined as in Formula (II).

A general method of preparing target molecules of Formula (II-B) byusing Intermediates 1f-2, 1g-2, 1h-2, and 1j-2 is outlined in Scheme2-2. Spiro-amines 1h-2 can be obtained via reduction of thiolactams 1g-2by treatment of 1f-2 with2,4-bis(4-methoxyphenyl)-2,4-dithioxo-1,3,2,4-dithiadiphosphetane(Lawesson reagent), followed by sodium borohydride (NaBH₄) in thepresence of, for instance, nickel (II) chloride hexahydrate(NiCl₂.6H₂O). Addition of the R-L moiety to achieve Intermediates 1j-2can be achieved via standard methods of alkylation, arylation,acylation, urea formation, or sulfonation. Treatment of 1j-2 withhydroxylamine and a base, e.g., NaOH, can provide compounds of Formula(II-B).

wherein L and R are defined as in Formula (II).

A general method of preparing target molecules of Formula (II-C) byusing Intermediates 1k-2, 1m-2, 1n-2, 1p-2, 1q-2, and 1r-2 is outlinedin Scheme 3-2. Spiro-amines 1p-2 can be obtained via the doubleα-alkylation of aryl bromide 1a-2. Double alkyl bromide amines 1n-2 canbe obtained from alcohols (1k-2) following protection with a benzylgroup 1m-2 followed by bromination under standard conditions (e.g.,treatment with PBr₃). Carbonylation of 1p-2 in the presence of a metalcatalyst, e.g., Pd(dppf)Cl₂, carbon monoxide, and a base, e.g., Et₃N,can provide esters 1q-2. Following debenzylation of 1q-2 under standardconditions (e.g., hydrogenation), to afford 1r-2, addition of the R-Lmoiety can be achieved via standard methods of alkylation, arylation,acylation, urea formation, or sulfonation to give 1s-2. Treatment of1s-2 with hydroxylamine and a base, e.g., NaOH, can provide compounds ofFormula (II-C).

wherein L and R are defined as in Formula (II).

Another general method of preparing target molecules of Formulae II-A(e.g., II-A1) and II-A2) by using Intermediates 1t-2, 1u-2, 1v-2, 1w-2,1x-2, 1y-2, 1z-2, 1aa-2, and 1bb-2 is outlined in Scheme 4-2. Doublebromination of 1-bromo-2,3-dimethylbenzene (1t-2), followed by in situgeneration of a reactive diene from 1u-2, for example, in the presenceof sodium iodide, to undergo a Diels-Alder cycloaddition with methylacrylate can afford a regioisomeric mixture of tetrahydronaphthalenes1v-2. Carbonylation of 1v-2 in the presence of a metal catalyst, e.g.,Pd(dppf)Cl₂, carbon monoxide, and a base, e.g., Et₃N, can provide esters1w-2, which can be alkylated by treatment with a halo-nitrile in thepresence of a base to provide Intermediates 1x-2. Reduction, forexample, with hydrogen gas in the presence of PtO₂, acetic acid, andMeOH, can be followed by treatment with NH₃ in MeOH to providespiro-lactams 1y-2 and 1aa-2, which can be separated by columnchromatography. Addition of the R-L moiety can be achieved via standardmethods of alkylation, acylation, urea formation, sulfonation orarylation. For example, alkylation of 1y-2 or 1aa-2 with an alkyl halidein the presence of a base, e.g., NaH, can provide compounds ofIntermediates 1z-2 and 1bb-2. Alternatively, arylation of 1y-2 or 1aa-2with an aryl boronic acid in the presence of a metal catalyst, e.g.,copper (II) acetate (Cu(OAc)₂), and a base, can also provide compoundsof Intermediates 1z-2 or 1bb-2. Treatment of 1z-2 or 1bb-2 withhydroxylamine and a base, e.g., NaOH, can provide compounds of Formulae(II-A1) and (II-A2).

wherein L and R are defined as in Formula (II).

Another general method of preparing target molecules of Formula II-C(e.g., II-C1) by using Intermediates 1 cc-2, 1dd-2, 1ee-2, 1ff-2, 1gg-2,and 1hh-2 is outlined in Scheme 5-2. Spirocycles 1ee-2 can be obtainedfrom the condensation of ketones 1cc-2 with ortho hydroxy-acetophenone1dd-2. Reduction, for example, with NaBH₄ in the presence of MeOH, canprovide alcohols 1ff-2. Dehydroxylation of 1ff-2 can be accomplished byconversion to the silyl ether using triethylsilane and treatment withTFA to provide Intermediates 1gg-2. Addition of the R-L moiety can beachieved via standard methods of alkylation, arylation, acylation, ureaformation, or sulfonation. Treatment of 1hh-2 with hydroxylamine and abase, e.g., NaOH, can provide compounds of Formula (II-C1).

wherein L and R are defined as in Formula (II).

Another general method of preparing target molecules of Formula II-C(e.g., II-C2) by using Intermediates 1cc-2, 1jj-2, 1kk-2, 1ll-2, 1mm-2,1nn-2, and 1oo-2 is outlined in Scheme 6-2. Spirocycles 1kk-2 can beobtained from the condensation of ketones 1cc-2 with orthohydroxy-acetophenone 1jj-2. Carbonylation of 1kk-2 in the presence of ametal catalyst, e.g., PdCl₂, carbon monoxide, and a diphosphine, canprovide esters 1ll-2. Reduction, for example, with NaBH₄ in the presenceof MeOH, can provide alcohols 1mm-2. Dehydroxylation of 1 mm-2 can beaccomplished by conversion to the silyl ether using triethylsilane andtreatment with TFA to provide Intermediates 1nn-2. Addition of the R-Lmoiety can be achieved via standard methods of alkylation, arylation,acylation, urea formation, or sulfonation. Treatment of 1oo-2 withhydroxylamine and a base, e.g., NaOH, can provide compounds of Formula(II-C2).

Methods of Using HDAC11 Inhibitors

The present disclosure provides methods of administering an effectiveamount of a therapeutic agent described herein (e.g., a HDAC11inhibitor) to a subject in need of treatment. In some embodiments areprovided methods of treating a disease associated with HDAC11 modulationin a subject in need thereof. In some embodiments, a method involvesadministering to a patient in need of treatment for diseases ordisorders associated with HDAC11 an effective amount of a HDAC11inhibitor. In some embodiments, a disease for treatment can be, but isnot limited to, a cell proliferative disease, a cancer, aneurodegenerative disease, a neurodevelopmental disease, an inflammatoryor autoimmune disease, an infection, a metabolic disease, a hematologicdisease, or a cardiovascular disease.

In some embodiments, a method of treating a cell proliferative diseasecomprises administering a HDAC11 inhibitor to a subject. In someembodiments, an effective amount (e.g., a therapeutic amount) of aHDAC11 inhibitor is administered.

In any of the methods described herein, a HDAC11 inhibitor of any classmay be used. In some embodiments, a HDAC11 inhibitor is a siRNA, ashRNA, an antibody agent, or a chemical compound (e.g., a smallmolecule). In some embodiments, a HDAC11 inhibitor is a siRNA, a shRNA,an antibody agent, or a chemical compound (e.g., a small molecule). Insome embodiments, a HDAC11 inhibitor is a chemical compound that is asmall molecule. In some embodiments, a small molecule HDAC11 inhibitoris at least 10-fold selective for the inhibition of HDAC11 over one ormore other histone deacetylase isoforms (e.g., HDAC1, HDAC2, HDAC3,HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9 and/or HDAC10). In someembodiments, a small molecule HDAC11 inhibitor is at least 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold,2,000-fold, 3,000-fold, or more selective for inhibition of HDAC11 overone or more other histone deacetylase isoforms (e.g., HDAC1, HDAC2,HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9 and/or HDAC10). In someembodiments, a small molecule HDAC11 inhibitor is at least 10-foldselective for the inhibition of HDAC11 over each of HDAC1, HDAC2, HDAC3,HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9 and HDAC10. In someembodiments, a HDAC11 inhibitor is specific for human HDAC11.

Cancers

In some embodiments are provided methods to treat cell proliferativediseases or disorders, such as, for example, cancer. Generally, in thecontext of the present disclosure, a cancer can be understood asabnormal or unregulated cell growth within a patient. In someembodiments, a cancer can include, but is not limited to, hematologicalmalignancy, lung cancer, ovarian cancer, breast cancer, prostate cancer,pancreatic cancer, hepatocellular cancer, renal cancer, melanoma, andleukemias such as acute myeloid leukemia and acute lymphoblasticleukemia. Additional cancer types include T-cell lymphoma (e.g.,cutaneous T-cell lymphoma, peripheral T-cell lymphoma), Hodgkinlymphoma, and multiple myeloma.

In some embodiments, the present disclosure encompasses the recognitionthat HDAC11 inhibitors may be beneficial for targeting cancer stem cells(CSCs). CSCs are cancer cells found within tumors or hematologicalcancers that possess characteristics associated with normal stem cells,specifically the ability to give rise to all cell types found in aparticular cancer sample. As a result, CSCs may generate tumors throughthe stem cell processes of self-renewal and differentiation and can thuspotentially persist in tumors causing relapse and metastasis by givingrise to new tumors. Specific therapies targeted at CSCs would bebeneficial for the treatment of patients, particularly those withmetastatic disease, however a challenge to development of therapiestargeted at CSCs is the identification of specific CSC markers to helpidentify therapies that can target certain CSCs specifically. In someembodiments, a HDAC11 inhibitor can specifically target CSCs. In someembodiments, a HDAC11 inhibitor may specifically target cancer cellsthat exhibit cancer stem cell properties and/or are associated with geneamplification of genes associated with cancer stem cell activity.

In some embodiments, the present disclosure provides a method oftreating a cancer wherein one or more cancer cells exhibit stemcell-like properties. In some embodiments, the cancer is associated witha gene amplification of SOX2. In some embodiments, the cancer associatedwith a gene amplification of SOX2 is esophageal squamous cell carcinoma,oral squamous cell carcinoma, lung, squamous cell carcinoma, lungadenocarcinoma, non-small cell lung cancer, small cell lung cancer orsinonasal cancer.

In some embodiments, the present disclosure provides methods forinhibiting expression of SOX2 in a stem-like cancer cell in a patienthaving a cancer, comprising administering a HDAC11 inhibitor to thepatient. Sex determining region Y-Box 2 (SOX2) is a gene that encodesfor a transcription factor belonging to the SOX gene family and has beendescribed as an essential embryonic stem cell gene and as a necessaryfactor for induced cellular reprogramming. Recent research has indicatedthat SOX2 is frequently overexpressed in a variety of human cancers andacts as an oncogene to confer certain stem cell properties to carcinomacells. Consequently, there has been a great deal of interest about therole of SOX2 in the clinic and potential therapeutic targets toselectively target SOX2-expressing cancer cells (Weina et al, Clin andTranslational Med. 2014, July; 3:19), however it has been challenging toidentify selective targets and therapies for SOX2-expressing cancercells. Accordingly, there remains a need for effective, safe andselective methods of treating diseases or disorders associated with geneamplification of SOX2. In some embodiments, a SOX2-expressing cancer islung cancer, hematological cancer or breast cancer. In some embodiments,a hematological cancer is MPN, lymphoma, or leukemia. In someembodiments, a cancer is esophageal squamous cell carcinoma, oralsquamous cell carcinoma, lung, squamous cell carcinoma, lungadenocarcinoma, non-small cell lung cancer, small cell lung cancer orsinonasal cancer.

In some embodiments, a cancer for treatment with a HDAC11 is a cancerthat is associated with a gene activation of STAT3. In some embodiments,a cancer associated with a gene activation of STAT3 is breast cancer.

In some embodiments, treating proliferative diseases or disorders caninclude any cancer where there is evidence of an increase inTreg/effector T cell ratio, and/or an increase in absolute Treg number,and/or increased expression of T cell tolerance-related genes. In someembodiments, an increase in Treg number can in the periphery, in thetumor microenvironment, and/or tertiary lymphoid structures. Suchproliferative diseases or disorders can include, but are not limited to:any Kras mutant carrying tumor(http://cancerimmunolres.aacrjournals.org/content/early/2016/02/13/2326-6066.CIR-15-0241.long);renal cell carcinoma; lung carcinoma; cervical cancer; prostate cancer;ovarian cancer; head and neck cancer; lymphoma; colorectal cancer,non-small cell lung carcinoma; breast cancers (Gobert, M. et al. (2009)Cancer Res. 69, 2000-2009); and bladder cancer.

In some embodiments, a cell proliferation disease is a hematologicalmalignancy. Hematological malignancies include, but are not limited to,leukemia (e.g., acute lymphocytic leukemia (ALL), acute myelogenousleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML), acute monocytic leukemia (AMoL)), lymphoma (e.g.,Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL)), and myeloma(e.g., multiple myeloma).

In some embodiments, a cell proliferation disease is a solid tumor.Examples of solid tumors include, but are not limited to, sarcomas,carcinomas, lung cancer, ovarian cancer, breast cancer, prostate cancer,pancreatic cancer, hepatocellular cancer, renal cancer, and melanoma. Insome embodiments, a solid tumor is benign. In some embodiments, a solidtumor is malignant.

In some embodiments, a cell proliferation disease is amyeloproliferative neoplasm (MPN). Myeloproliferative neoplasms arehematological diseases characterized by excessive and chronic productionof mature cells from one or several myeloid lineages (Sonbol et al, TherAdv Hematol. 2013, February; 4(1):15-35). MPNs include, but are notlimited to, polycythemia vera (PV), essential thrombocythemia (ET),primary myelofibrosis (PMF), chronic myelogenous leukemia (CML), chronicneutrophilic leukemia (CNL), and chronic eosinophilic leukemia (CEL).

MPNs are often associated with mutations in JAK2 or MPL. Currenttreatments for MPNs include the use of Janus kinase 2 (JAK2) inhibitorssuch as ruxolitinib; however, some cells remain refractory to treatmentwith JAK2 inhibitors. Moreover, current therapies treat the symptomsrather than the disease. While class I and/or class II HDAC inhibitorshave been suggested for treating certain MPNs (WO 2010/034693, US2011/0237663, and US 2014/0039059), pan-HDAC inhibitors can result incellular toxicity due to the large role HDACs play in regulatingtranscription. Accordingly, there remains a need for effective, safe,and selective methods of treating cell proliferative diseases,particularly MPNs.

In certain embodiments, a MPN for treatment with a HDAC11 inhibitor isselected from PV, ET, and PMF. In certain embodiments, a MPN is PV. Incertain embodiments, a MPN is ET. In certain embodiments, a MPN is PMF.

Inflammatory and Autoimmune Diseases

The present disclosure encompasses the recognition that inhibition ofHDAC11 may be beneficial for treatment or prevention of inflammatoryand/or autoimmune diseases. Without wishing to be bound to theory, it isenvisioned that inhibition of HDAC11 may exert effects on immune cellregulation (e.g., T cells, B cells, macrophages, and/or antigenpresenting cells (APC)). For example, the protein interactome of HDAC11in T cells suggests a wide variety of substrates and biologicalprocesses (Joshi et al, Mol Syst Biol. 2013, 9:672). HDAC11 alsoregulates interleukin 10 (IL-10) expression in antigen presenting cellsand myeloid-derived suppressor cells (Villagra et al, Nat Immunol. 2009,January; 10(1):92-100; Cheng et al, Mol Immunol. 2014, July;60(1):44-53; Sahakian et al, Mol Immunol. 2015, February; 63(2):579-85).HDAC11 also plays a role in regulating OX40 ligand in antigen presentingcells (Buglio et al, Blood. 2011, Mar. 10; 117(10):2910-7).

In some embodiments, a disease or disorder for treatment with a HDAC11inhibitor is an immune or inflammatory disorder, which may be acute orchronic. Examples of immune and inflammatory disorders includeinflammatory bowel disease, psoriasis, rheumatoid arthritis, multiplesclerosis, and Alzheimer's disease.

Combination Therapies

A HDAC11 inhibitor may be administered alone or in combination withother drugs (e.g., as an adjuvant). For example, one or more HDAC11inhibitors can be in combination with other agents for treatment orprevention of diseases disclosed herein.

In some embodiments, a HDAC11 inhibitor is administered in combinationwith at least one other anticancer agent, including, for example, anychemotherapeutic agent known in the art, ionizing radiation, smallmolecule anticancer agents, surgery and/or biological therapies (e.g.,antibody agents, viruses, gene therapies, cell-based therapies, etc.).

In some embodiments, a method for inhibiting proliferation of aneoplastic cell in a patient having a MPN comprising administering acombination of a HDAC11 inhibitor and a JAK2 inhibitor to the patient.In some embodiments, a HDAC11 inhibitor and JAK2 inhibitor areco-administered. In another embodiment, the HDAC11 inhibitor and JAK2inhibitor are administered separately. In some embodiments, a HDAC11inhibitor and JAK2 inhibitor are administered in series. In someembodiments, a HDAC11 inhibitor is administered prior to the JAK2inhibitor. In another embodiment, the JAK2 inhibitor is administeredprior to the HDAC11 inhibitor. In some embodiments, a MPN is PV, ETand/or MPF. In some embodiments, a HDAC11 inhibitor is a polypeptide, apolynucleotide, a siRNA, a shRNA, an antibody agent, or a chemicalcompound. In some embodiments, a JAK2 inhibitor is a polypeptide, apolynucleotide, a siRNA, a shRNA, an antibody agent, or a chemicalcompound. In some embodiment, a JAK2 inhibitor is ruxolitinib,baricitinib, CYT387, lestaurtinib, or pacritinib. In some embodiments, aJAK2 inhibitor is JAK2 inhibitor is ruxolitinib, baricitinib, CYT387,lestaurtinib, or pacritinib.

In some embodiments, the present disclosure provides a method forreducing tumor burden in a patient having a myeloproliferative neoplasm,comprising administering a combination of a HDAC11 inhibitor and a JAK2inhibitor to the patient. In some embodiments, a HDAC11 inhibitor andJAK2 inhibitor are co-administered. In another embodiment, the HDAC11inhibitor and JAK2 inhibitor are administered separately. In someembodiments, a HDAC11 inhibitor and JAK2 inhibitor are administered inseries. In some embodiments, a HDAC11 inhibitor is administered prior tothe JAK2 inhibitor. In another embodiment, a JAK2 inhibitor isadministered prior to the HDAC11 inhibitor. In some embodiments, aHDAC11 inhibitor is administered to a patient that has been or will beadministered a JAK2 inhibitor, such that the patient receives treatmentwith both. In some embodiments, a JAK2 inhibitor is administered to apatient that has been or will be administered a HDAC11 inhibitor, suchthat the patient receives treatment with both. In some embodiments, aMPN is PV, ET and/or MPF. In some embodiments, a HDAC11 inhibitor is apolypeptide, a polynucleotide, a siRNA, a shRNA, an antibody agent, or achemical compound. In some embodiments, a JAK2 inhibitor is apolypeptide, a polynucleotide, a siRNA, a shRNA, an antibody agent, or achemical compound. In some embodiments, a JAK2 inhibitor is JAK2inhibitor is ruxolitinib, baricitinib, CYT387, lestaurtinib, orpacritinib. In some embodiments, a JAK2 inhibitor is ruxolitinib.

In some embodiments, the present disclosure provides a method forinhibiting proliferation of a cell resistant to a JAK2 inhibitor in apatient having a MPN, comprising administering a HDAC11 inhibitor to thepatient. In some embodiments, a MPN is PV, ET and/or MPF. In someembodiments, a HDAC11 inhibitor is a polypeptide, a polynucleotide, asiRNA, a shRNA, an antibody agent, or a chemical compound. In someembodiments, a JAK2 inhibitor is a polypeptide, a polynucleotide, asiRNA, a shRNA, an antibody agent, or a chemical compound. In someembodiments, a JAK2 inhibitor is ruxolitinib, baricitinib, CYT387,lestaurtinib, or pacritinib. In some embodiments, a JAK2 inhibitor isruxolitinib.

In some embodiments, the present disclosure provides a method forinducing cell cycle arrest in a patient having a MPN, comprisingadministering a combination of a HDAC11 inhibitor and a JAK2 inhibitorto the patient. In some embodiments, a HDAC11 inhibitor and a JAK2inhibitor are co-administered. In other embodiments, a HDAC11 inhibitorand a JAK2 inhibitor are administered separately. In some embodiments, aHDAC11 inhibitor and a JAK2 inhibitor are administered in series. Insome embodiments, a HDAC11 inhibitor is administered prior toadministration of a JAK2 inhibitor. In other embodiments, a JAK2inhibitor is administered prior to a HDAC11 inhibitor. In someembodiments, a HDAC11 inhibitor is administered to a patient that hasbeen or will be administered a JAK2 inhibitor, such that the patientreceives treatment with both. In some embodiments, a JAK2 inhibitor isadministered to a patient that has been or will be administered a HDAC11inhibitor, such that the patient receives treatment with both.

In some embodiments, a MPN is PV, ET and/or MPF. In some embodiments, aHDAC11 inhibitor is a polypeptide, a polynucleotide, a siRNA, a shRNA,an antibody agent, or a chemical compound. In some embodiments, a JAK2inhibitor is a polypeptide, a polynucleotide, a siRNA, a shRNA, anantibody agent, or a chemical compound. In some embodiments, a JAK2inhibitor is ruxolitinib, baricitinib, CYT387, lestaurtinib, orpacritinib. In some embodiments, a JAK2 inhibitor is ruxolitinib. Insome embodiments, a cell cycle is arrested at G1.

In some embodiments, the HDAC11 inhibitor is a polypeptide, apolynucleotide, a siRNA, a shRNA, an antibody agent, or a chemicalcompound. In some embodiments, the method further comprisesadministering a JAK2 inhibitor. In some embodiments, the JAK2 inhibitoris a polypeptide, a polynucleotide, a siRNA, a shRNA, an antibody agent,or a chemical compound. In some embodiments, the JAK2 inhibitor isruxolitinib, baricitinib, CYT387, lestaurtinib, or pacritinib. In someembodiments, the JAK2 inhibitor is ruxolitinib.

In some embodiments, a method further comprises administering a hedgehogpathway inhibitor. In some embodiments, the hedgehog pathway inhibitoris vismodegib, erismodegib, BMS-833923, glasdegib, taladegib, orsaridegib. In some embodiments, a HDAC11 inhibitor is administered to apatient that has been or will be administered a hedgehog pathwayinhibitor, such that the patient receives treatment with both. In someembodiments, a hedgehog pathway inhibitor is administered to a patientthat has been or will be administered a HDAC11 inhibitor, such that thepatient receives treatment with both.

In some embodiments, a method for inhibiting expression of SOX2 in astem-like cancer cell in a patient in need thereof are provided,comprising administering a HDAC11 inhibitor in combination with one ormore additional therapeutic agents. In some embodiments, a cancer islung cancer, hematological cancer or breast cancer. In some embodiments,a hematological cancer is MPN, lymphoma, or leukemia. In someembodiments, a cancer is esophageal squamous cell carcinoma, oralsquamous cell carcinoma, lung, squamous cell carcinoma, lungadenocarcinoma, non-small cell lung cancer, small cell lung cancer orsinonasal cancer. In some embodiments, a HDAC11 inhibitor is apolypeptide, a polynucleotide, a siRNA, a shRNA, an antibody agent, or achemical compound.

In some embodiments, a method for inhibiting expression of SOX2 in astem-like cancer cell in a patient in thereof comprises administering aHDAC11 inhibitor in combination with a JAK2 inhibitor. In someembodiments, a JAK2 inhibitor is a polypeptide, a polynucleotide, asiRNA, a shRNA, an antibody agent, or a chemical compound. In someembodiments, a JAK2 inhibitor is ruxolitinib, baricitinib, CYT387,lestaurtinib, or pacritinib. In some embodiments, a JAK2 inhibitor isruxolitinib.

In some embodiments, a method for inhibiting expression of SOX2 in astem-like cancer cell in a patient in thereof comprises administering aHDAC11 inhibitor in combination with a hedgehog pathway inhibitor. Insome embodiments, a hedgehog pathway inhibitor is vismodegib,erismodegib, BMS-833923, glasdegib, taladegib, or saridegib.

In another embodiment, methods of treating cancer wherein one or morecancer cells exhibit stem cell-like properties are provided comprisingtreating a patient with a first line therapy; and administering to thepatient a HDAC11 inhibitor, whereby any cancer cells surviving from thefirst line therapy are reduced or eliminated after treatment with theHDAC11 inhibitor. In some embodiments, the cancer is esophageal squamouscell carcinoma, oral squamous cell carcinoma, lung squamous cellcarcinoma, lung adenocarcinoma, non-small cell lung cancer, small celllung cancer or sinonasal cancer. In other embodiments, the cancer isbreast cancer. In some embodiments, the HDAC11 inhibitor is a siRNA, ashRNA, an antibody agent, or a chemical compound. In other embodiments,the chemical compound is a small molecule that is at least 10-foldselective for the inhibition of HDAC11 over other histone deacetylaseisoforms. In some embodiments, the first line therapy is resection,radiation, or stem cell transplant.

Formulation and Administration

Administration of therapeutic agents encompassed by the presentdisclosure (e.g., a HDAC11 inhibitor and/or a JAK2 inhibitor or ahedgehog pathway inhibitor) can be accomplished via any mode ofadministration for therapeutic agents. These modes include systemic orlocal administration such as oral, nasal, parenteral, transdermal,subcutaneous, vaginal, buccal, rectal or topical administration modes.

Depending on the intended mode of administration, therapeutic agentsdescribed herein can be in solid, semi-solid or liquid dosage form, suchas, for example, injectables, tablets, suppositories, pills,time-release capsules, elixirs, tinctures, emulsions, syrups, powders,liquids, suspensions, or the like, sometimes in unit dosages andconsistent with conventional pharmaceutical practices. Likewise, theycan also be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous or intramuscular form, all using formswell known to those skilled in the pharmaceutical arts.

Illustrative pharmaceutical compositions are tablets and gelatincapsules comprising a therapeutic agent and a pharmaceuticallyacceptable carrier, such as a) a diluent, e.g., purified water,triglyceride oils, such as hydrogenated or partially hydrogenatedvegetable oil, or mixtures thereof, corn oil, olive oil, sunflower oil,safflower oil, fish oils, such as EPA or DHA, or their esters ortriglycerides or mixtures thereof, omega-3 fatty acids or derivativesthereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose,sodium, saccharin, glucose and/or glycine; b) a lubricant, e.g., silica,talcum, stearic acid, its magnesium or calcium salt, sodium oleate,sodium stearate, magnesium stearate, sodium benzoate, sodium acetate,sodium chloride and/or polyethylene glycol; for tablets also; c) abinder, e.g., magnesium aluminum silicate, starch paste, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesiumcarbonate, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth orsodium alginate, waxes and/or polyvinylpyrrolidone, if desired; d) adisintegrant, e.g., starches, agar, methyl cellulose, bentonite, xanthangum, algiic acid or its sodium salt, or effervescent mixtures; e)absorbent, colorant, flavorant and sweetener; f) an emulsifier ordispersing agent, such as Tween 80, Labrasol, HPMC, DOSS, caproyl 909,labrafac, labrafil, peceol, transcutol, capmul MCM, capmul PG-12, captex355, gelucire, vitamin E TGPS or other acceptable emulsifier; and/or g)an agent that enhances absorption of the compound such as cyclodextrin,hydroxypropyl-cyclodextrin, PEG400, PEG200.

Liquid, particularly injectable, compositions can, for example, beprepared by dissolution, dispersion, etc. For example, the disclosedcompound is dissolved in or mixed with a pharmaceutically acceptablesolvent such as, for example, water, saline, aqueous dextrose, glycerol,ethanol, and the like, to thereby form an injectable isotonic solutionor suspension. Proteins such as albumin, chylomicron particles, or serumproteins can be used to solubilize the disclosed compounds.

Therapeutic agents can be also formulated as a suppository that can beprepared from fatty emulsions or suspensions; using polyalkylene glycolssuch as propylene glycol, as the carrier.

Therapeutic agents can also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, containing cholesterol, stearylamine orphosphatidylcholines. In some embodiments, a film of lipid components ishydrated with an aqueous solution of drug to a form lipid layerencapsulating the drug, as described in U.S. Pat. No. 5,262,564.

Therapeutic agents can also be delivered by the use of monoclonalantibodies as individual carriers to which the disclosed compounds arecoupled. The disclosed compounds can also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the disclosedcompounds can be coupled to a class of biodegradable polymers useful inachieving controlled release of a drug, for example, polylactic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked oramphipathic block copolymers of hydrogels. In some embodiments,disclosed compounds are not covalently bound to a polymer, e.g., apolycarboxylic acid polymer, or a polyacrylate.

Parental injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions or solid forms suitable for dissolving in liquid prior toinjection.

The present disclosure provides pharmaceutical compositions comprising atherapeutic agent (e.g., HDAC11 inhibitor) and a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carrier can furtherinclude an excipient, diluent, or surfactant. The composition, ifdesired, can also contain one or more additional therapeutically activesubstances.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation.

Compositions can be prepared according to conventional mixing,granulating or coating methods, respectively, and the presentpharmaceutical compositions can contain from about 0.1% to about 99%,from about 5% to about 90%, or from about 1% to about 20% of atherapeutic agent described herein by weight or volume.

A dosage regimen utilizing the therapeutic agent is selected inaccordance with a variety of factors including type, species, age,weight, sex and medical condition of the patient; the severity of thecondition to be treated; the route of administration; the renal orhepatic function of the patient; and the particular therapeutic agentemployed. A physician or veterinarian of ordinary skill in the art canreadily determine and prescribe the effective amount of the therapeuticagent required to prevent, counter or arrest the progress of thecondition.

Effective dosage amounts of therapeutic agents, when used for theindicated effects, range from about 0.5 mg to about 5000 mg of thedisclosed compound as needed to treat the condition. Compositions for invivo or in vitro use can contain about 0.5, 5, 20, 50, 75, 100, 150,250, 500, 750, 1000, 1250, 2500, 3500, or 5000 mg of the therapeuticagent, or, in a range of from one amount to another amount in the listof doses. In some embodiments, compositions are in the form of a tabletthat can be scored.

Kits

The present disclosure further provides a pharmaceutical pack or kitcomprising one or more containers filled with at least one HDAC11inhibitor as described herein. Kits may be used in any applicablemethod, including, for example, therapeutically, diagnostically, etc.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflects(a) approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

In some embodiments, a kit may include one or more reagents fordetection (e.g, detection of a HDAC11 inhibitor). In some embodiments, akit may include a HDAC11 inhibitor in a detectable form (e.g.,covalently associated with detectable moiety or entity).

In some embodiments, a HDAC11 inhibitor as provided herein may beincluded in a kit used for detection and/or treatment of subjects.

The following numbered embodiments, while non-limiting, are exemplary ofcertain aspects of the present disclosure:

-   1. A compound of the Formula I:

-   -   and pharmaceutically acceptable salts thereof, wherein:        -   Q is —H, —OC(O)NR⁶(C₁-C₆)alkylaryl, or            —OC(O)O(C₁-C₆)alkylaryl        -   Z is —CH₂—, O, S or NR⁶;        -   X₁, X₂, X₃, and X₄ are each independently, at each            occurrence, N or CR¹;        -   Y₁, Y₂, and Y₃ are each independently N or CR¹;        -   L is NR⁶, O, or —(CR¹R²)_(p)—;        -   R¹ and R² are independently, at each occurrence, —H, —R³,            —R⁴, —C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl,            —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,            heteroaryl containing 1-5 heteroatoms selected from the            group consisting of N, S, P and O, —OH, —OR³′ halogen, —NO₂,            —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂, —S(O)₂NH₂,            —S(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)₂R⁵,            —S(O)₂(C₁-C₆alkyl), —(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl,            —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, or            —(CHR⁵)_(p)NR³R⁴, wherein each alkyl, alkenyl, cycloalkenyl,            alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is            optionally substituted with one or more —OH, halogen, —NO₂,            Oxo, —CN, —R³, —R⁵, —SR³, —OR³, —NHR³, —NR³R⁴,            —S(O)₂N(R³)₂—, —S(O)₂R⁵, —C(O)R⁵, —C(O)OR⁵, —NR³S(O)₂R⁵,            —S(O)R⁵, —S(O)NR³R⁴, —NR³S(O)R⁵, heterocycle, aryl, or            heteroaryl;        -   or R¹ and R² can combine with the carbon atom to which they            are both attached to form a spirocycle, spiroheterocycle, or            spirocycloalkenyl, each optionally substituted with one or            more independent occurrences of R³ and R⁴;        -   or two occurrences of R¹, when on adjacent atoms, can            combine to form a cycloalkyl, a heterocycle, aryl,            heteroaryl containing 1-5 heteroatoms selected from the            group consisting of N, S, P and O, or a cycloalkenyl, each            optionally substituted with one or more independent            occurrences of R³ and R⁴;        -   R³ and R⁴ are independently, at each occurrence, —H,            —C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl,            —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,            heteroaryl containing 1-5 heteroatoms selected from N, S, P,            and O, —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂(C₁-C₆alkyl),            —(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, or            —(CHR⁵)_(p)N(C₁-C₆alkyl)₂, wherein each alkyl, alkenyl,            cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and            heteroaryl is optionally substituted with one or more            substituents selected from —OH, halogen, —NO₂, oxo, —CN,            —R⁵, —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl, —N(C₁-C₆alkly)₂,            —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl,            —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵,            —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle,            aryl, or heteroaryl;        -   R⁵ is independently, at each occurrence, —H, —C₁-C₆alkyl,            —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,            —C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing            1-5 heteroatoms selected from N, S, P and O, —OH, halogen,            —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,            —S(O)₂NH(C₁-C₆alkyl), —S(O)₂N(C₁-C₆alkyl)₂,            —S(O)₂C₁-C₆alkyl, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,            —N(C₁-C₆alkyl)SO₂C₁-C₆alkyl, —S(O)(C₁-C₆alkyl),            —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)(C₁-C₆alkyl) or            —(CH₂)_(p)N(C₁-C₆alkyl)₂;        -   R⁶ is independently, at each occurrence, —H, —C₁-C₆alkyl,            —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,            —C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing            1-5 heteroatoms selected from the group consisting of N, S,            P and O, —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂(C₁-C₆alkyl),            —(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, or            —(CHR⁵)_(p)NR³R⁴ wherein each alkyl, alkenyl, cycloalkenyl,            alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is            optionally substituted with one or more substituents            selected from —OH, halogen, —NO₂, oxo, —CN, —R⁵,            —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl, —N(C₁-C₆alkyl)₂,            —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl,            —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵,            —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle,            aryl, or heteroaryl; and        -   p is 0, 1, 2, 3, 4, 5, or 6.

-   2. The compound of embodiment 1, wherein one of Y₁, Y₂ or Y₃ is N    and the other two of Y₁, Y₂ or Y₃ are each independently CR.

-   3. The compound of embodiment 1, wherein two of Y₁, Y₂ or Y₃ are N    and the other one of Y₁, Y₂ or Y₃ is CR¹.

-   4. The compound of embodiment 2 or 3, wherein R¹ is H.

-   5. The compound of embodiment 1, wherein L is S.

-   6. The compound of embodiment 1, wherein L is O.

-   7. The compound of embodiment 1, wherein L is NR⁶.

-   8. The compound of embodiment 1, wherein the compound is represented    by Formula I-A:

-   9. The compound of embodiment 1, wherein the compound is represented    by Formula I-B:

-   10. The compound of embodiment 1, wherein the compound is    represented by Formula I-C:

-   11. The compound of embodiment 1, 8, 9 or 10, wherein X₁ and X₄ are    both N, and X₂ and X₃ are each independently CR¹.-   12. The compound of embodiment 1, 8, 9 or 10, wherein X₂ and X₄ are    both N, and X₁ and X₃ are each independently CR¹.-   13. The compound of embodiment 1, 8, 9 or 10, wherein X₁ is N and    X₂, X₃ and X₄ are each independently CR¹.-   14. The compound of embodiment 1, 8, 9 or 10, wherein each    occurrence of R⁶ is independently H, —C₁-C₆alkyl,    —(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, or    —(CHR⁵)_(p)NR³R⁴ wherein each alkyl is optionally substituted with    one or more substituents selected from —OH, halogen, —NO₂, oxo, —CN,    —R⁵, —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl, —N(C₁-C₆alkyl)₂,    —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl,    —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵,    —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, or    heteroaryl.-   15. The compound of embodiment 14, wherein each occurrence of R⁶ is    independently H, —(CHR⁵)_(p)NR³R⁴, or —C₁-C₆alkyl optionally    substituted with one or more substituents selected from —OH,    halogen, —NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl,    —N(C₁-C₆alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl,    —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl,    —S(O)R⁵, —S(O)N(C₁-C₆alkyl)₂, or —N(C₁-C₆alkyl)S(O)R⁵.-   16. The compound of embodiment 1, 8, 9 or 10, wherein each    independent occurrence of R¹ is halogen, —CF₃, —OH, —CN,    —SO₂(C₁-C₃alkyl), phenyl, C₁-C₃alkyl, C₁-C₃alkoxy, pyridyl,    —C(O)C₁-C₃alkyl, —OC₁-C₃alkyl, —(C₁-C₃alkyl)O(C₁-C₃alkyl), —OCF₃ or    —OCH₂phenyl.-   17. A compound of the Formula II:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   X¹, X², X³, X⁴, X⁵, and X⁶ are each independently, at each        occurrence, —CR¹R²—, —NR³—, —O—, —C(O)—, —S(O)₂—, —S(O)—, or        —S—;    -   Y¹, Y², and Y³ are each independently N or CR¹;    -   L is a bond, —(CR¹R²)_(p)—, —C(O)NR³—, —S(O)₂—, —S(O)₂NR³—,        —S(O)—, —S(O)NR³—, —C(O)(CR¹R²)_(p)O—, or —C(O)(CR¹R²)_(p)—;    -   R is independently —H, —C₁-C₆alkyl, —C₂-C₆alkenyl,        —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl,        —C₅-C₁₂spirocycle, heterocyclyl, spiroheterocyclyl, aryl, or        heteroaryl containing 1-5 heteroatoms selected from the group        consisting of N, S, P, or O, wherein each alkyl, alkenyl,        cycloalkenyl, alkynyl, cycloalkyl, spirocycle, heterocyclyl,        spiroheterocyclyl, aryl, or heteroaryl is optionally substituted        with one or more —OH, halogen, oxo, —NO₂, —CN, —R¹, —R², —SR³,        —OR³, —NHR³, —NR³R⁴, —S(O)₂NR³R⁴, —S(O)₂R¹, —C(O)R¹, —C(O)OR¹,        —NR³S(O)₂R¹, —S(O)R¹, —S(O)NR³R⁴, —NR³S(O)R¹, heterocycle, aryl,        or heteroaryl;    -   R¹ and R² are independently, at each occurrence, —H, —R³, —R⁴,        —C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,        —C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5        heteroatoms selected from the group consisting of N, S, P and O,        —OH, halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,        —S(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)₂R⁵, —S(O)₂(C₁-C₆alkyl),        —(C₁-C₆alkyl)S(O)₂R, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,        —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, or —(CHR⁵)_(p)NR³R⁴, wherein each        alkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl,        aryl, or heteroaryl is optionally substituted with one or more        —OH, halogen, —NO₂, oxo, —CN, —R⁵, —OR³, —NHR³, —NR³R⁴,        —S(O)₂N(R³)₂—, —S(O)₂R⁵, —C(O)R⁵, —C(O)OR⁵, —NR³S(O)₂R⁵,        —S(O)R⁵, —S(O)NR³R⁴, —NR³S(O)R⁵, heterocycle, aryl, or        heteroaryl;    -   or R¹ and R² can combine with the carbon atom to which they are        both attached to form a spirocycle, spiroheterocycle, or        spirocycloalkenyl;    -   or R¹ and R², when on adjacent atoms, can combine to form a        cycloalkyl, cycloalkenyl, heterocycle, aryl, or heteroaryl        containing 1-5 heteroatoms selected from the group consisting of        N, S, P and O;    -   or R¹ and R², when on non-adjacent atoms, can combine to form an        optionally bridging cycloalkyl, an optionally bridging        heterocycle, or an optionally bridging cycloalkenyl;    -   R³ and R⁴ are independently, at each occurrence, —H,        —C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,        —C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5        heteroatoms selected from N, S, P, and O, —S(O)₂N(C₁-C₆alkyl)₂,        —S(O)₂(C₁-C₆alkyl), —(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl,        —C(O)OC₁-C₆alkyl, or —(CHR⁵)_(p)N(C₁-C₆alkyl)₂, wherein each        alkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl,        aryl, and heteroaryl is optionally substituted with one or more        substituents selected from —OH, halogen, —NO₂, oxo, —CN, —R⁵,        —O(C₁-C₆)alkyl, —NH(C₁-C₆)alkyl, —N(C₁-C₆alkly)₂,        —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl,        —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵,        —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, or        heteroaryl;    -   R⁵ is independently, at each occurrence, —H, —C₁-C₆alkyl,        —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,        —C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5        heteroatoms selected from N, S, P and O, —OH, halogen, —NO₂,        —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂, —S(O)₂NH(C₁-C₆alkyl),        —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂C₁-C₆alkyl, —C(O)C₁-C₆alkyl,        —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)SO₂C₁-C₆alkyl,        —S(O)(C₁-C₆alkyl), —S(O)N(C₁-C₆alkyl)₂,        —N(C₁-C₆alkyl)S(O)(C₁-C₆alkyl) or —(CH₂)_(p)N(C₁-C₆alkyl)₂;    -   p is 0, 1, 2, 3, 4, 5, or 6;    -   n is 0, 1, 2, 3, or 4;    -   m is 0, 1, or 2;    -   q is 1 or 2; and    -   r is 1 or 2;    -   wherein the sum q+r≤3 and    -   wherein the sum m+n≤4.

-   18. The compound of embodiment 17, wherein n is 0 and m is 1.

-   19. The compound of embodiment 17, wherein n is 1 and m is 1.

-   20. The compound of embodiment 17, wherein q is 1 and r is 1.

-   21. The compound of embodiment 17, wherein q is 2 and r is 1.

-   22. The compound of embodiment 17, wherein q is 1 and r is 2.

-   23. The compound of embodiment 17, wherein q is 1, r is 1, m is 0    and n is 1.

-   24. The compound of embodiment 17, wherein q is 1, r is 1, m is 1    and n is 1.

-   25. The compound of embodiment 17, wherein q is 1, r is 1, m is 2    and n is 1.

-   26. The compound of embodiment 17, wherein q is 1, r is 1, m is 1    and n is 2.

-   27. The compound of embodiment 17, wherein X⁵ is C(O).

-   28. The compound of embodiment 17, wherein X⁴ is C(O).

-   29. The compound of embodiment 17, wherein the compound is of the    Formula II-A:

-   30. The compound of embodiment 17, wherein the compound is of the    Formula II-B:

-   31. The compound of embodiment 17, wherein the compound is of the    Formula II-C:

-   32. The compound of embodiment 17, wherein the compound is of the    Formula II-D:

-   33. The compound of embodiment 17, wherein the compound is of the    Formula II-E:

-   34. The compound of any one of embodiments 29-33, wherein L is    selected from a bond, —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —C(O)—, —S(O)₂—,    —C(O)NR³—, or —C(O)CH₂—.-   35. The compound of any one of embodiments 29-33, wherein L is    selected from —CH₂—, or —C(O)—.-   36. The compound of any one of embodiments 29-33, wherein R is    hydrogen or an optionally substituted group selected from    C₁-C₆alkyl, aryl, C₃-C₈ cycloalkyl, heterocyclyl, or heteroaryl    containing 1-5 heteroatoms selected from the group consisting of N,    S, P, or O.-   37. The compound of any one of embodiments 29-33, wherein R is    hydrogen or an optionally substituted group selected from phenyl,    C₁-C₆alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,    pyridyl, or morpholinyl.-   38. The compound of any one of embodiments 29-33, wherein R is    phenyl optionally substituted with one or more independent    occurrences of halogen, CF₃, —SO₂CH₃, phenyl, C₁-C₃ alkyl,    C₁-C₃alkoxy, pyridyl, —OCF₃ or —OCH₂phenyl.-   39. The compound of embodiment 17, wherein the compound is of the    Formula II-A-i:

-   40. The compound of embodiment 17, wherein the compound is of the    Formula II-B-i:

-   41. The compound of embodiment 17, wherein the compound is of the    Formula II-C-i or II-C-ii:

-   42. The compound of embodiment 17, wherein the compound is of the    Formula II-D-i:

-   43. The compound of embodiment 17, wherein the compound is of the    Formula II-E-i:

-   44. The compound of any one of embodiments 39-43, wherein L is    selected from a bond, —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —C(O)—, —S(O)₂—,    —C(O)NR³—, or —C(O)CH₂—.-   45. The compound of any one of embodiments 39-43, wherein L is    selected from —CH₂—, or —C(O)—.-   46. The compound of any one of embodiments 39-43, wherein R is    hydrogen or an optionally substituted group selected from    C₁-C₆alkyl, aryl, C₃-C₈ cycloalkyl, heterocyclyl, or heteroaryl    containing 1-5 heteroatoms selected from the group consisting of N,    S, P, or O.-   47. The compound of any one of embodiments 39-43, wherein R is    hydrogen or an optionally substituted group selected from phenyl,    C₁-C₆alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,    pyridyl, or morpholinyl.-   48. The compound of any one of embodiments 39-43, wherein R is    phenyl optionally substituted with one or more independent    occurrences of halogen, CF₃, —SO₂CH₃, phenyl, C₁-C₃ alkyl,    C₁-C₃alkoxy, pyridyl, —OCF₃ or —OCH₂phenyl.

EXAMPLES

The disclosure is further illustrated by the following examples, whichare not to be construed as limiting this disclosure in scope or spiritto the specific procedures herein described. It is to be understood thatthe examples are provided to illustrate certain embodiments and that nolimitation to the scope of the disclosure is intended thereby. It is tobe further understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which may suggestthemselves to those skilled in the art without departing from the spiritof the present disclosure and/or scope of the appended claims.

Certain abbreviations used in the following Examples and elsewhereherein:

ACN acetonitrile (CH₃CN)AcOH acetic acidAIBN 2,2′-Azobis(2-methylpropionitrile)CH₃CN acetonitrileDCE 1,2-dichloroethaneDCM dichloromethane or methylene chloride

DEA N,N-diethylamine DIEA N,N-diisopropylethylamine DMAN,N-Dimethylacetamide DMF N,N-dimethylformamide

DMSO dimethylsulfoxideDMTMM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloridedppf bis(diphenylphosphino)ferroceneEtOAc ethyl acetateEtOH ethanolFA formic acidh hoursHATU2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphateHBr hydrogen bromideHCl hydrogen chlorideHPLC high performance liquid chromatographyLC/MS liquid chromatography/mass spectrometryLiOH lithium hydroxideK₂CO₃ potassium carbonateMeOH methanolMS mass spectrometryNaOH sodium hydroxideNa₂SO₄ sodium sulfateNH₄HCO₃ ammonium bicarbonateNMM 4-methylmorpholineNMP N-Methyl-2-pyrrolidonePd₂(dba)₃ tris(dibenzylideneacetone)dipalladium

Pd(dppf)Cl₂ [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (II)

PMB para-methoxybenzylPPh₃ triphenylphosphinert room temperatureRuPhos 2-Dicyclohexylphosphino-2′,6′-diisopropoxybiphenylRuPhos 2GChloro(2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II),TEA triethylamineTFA trifluoroacetic acidTHF tetrahydrofuranXantPhos 4,5-Bis(diphenylphosphino)-9,9-dimethylxantheneXPhos 2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl XPhos 2GChloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)XPhos 3GMethanesulfonato(2-dicyclohexylphosphino-2′,4′,6′-tri-i-propyl-1,1′-biphenyl)(2′-amino-1,1′-biphenyl-2-yl)palladium(II)dichloromethane adduct

Materials

Unless otherwise noted, all materials were obtained from commercialsuppliers and were used without further purification. Anhydrous solventswere obtained from Sigma-Aldrich (Milwaukee, Wis.) and used directly.All reactions involving air- or moisture-sensitive reagents wereperformed under a nitrogen atmosphere.

Unless otherwise noted, mass-triggered HPLC purification and/or purityand low resolution mass spectral data were measured using either: (1)Waters Acquity ultra performance liquid chromatography (UPLC) system(Waters Acquity UPLC with Sample Organizer and Waters Micromass ZQ MassSpectrometer) with UV detection at 220 nm and a low resonanceelectrospray positive ion mode (ESI) (Column: Acquity UPLC BEH C18 1.7μm 2.1×50 mm; gradient: 5-100% Solvent B (95/5/0.09%:Acetonitrile/Water/Formic Acid) in Solvent A (95/5/0.1%: 10 mM AmmoniumFormate/Acetonitrile/Formic Acid) for 2.2 min then 100-5% Solvent B inSolvent A for 0.01 min then hold at 5% Solvent B in Solvent A for 0.29min) or (2) Waters HT2790 Alliance high performance liquidchromatography (HPLC) system (Waters 996 PDA and Waters ZQ Single QuadMass Spectrometer) with UV detection at 220 nm and 254 nm and a lowresonance electrospray ionization (positive/negative) mode (ESI)(Column: XBridge Phenyl or C18, 5 μm 4.6×50 mm; gradient: 5-95% SolventB (95% methanol/5% water with 0.1% Formic Acid) in Solvent A (95%water/5% methanol with 0.1% Formic Acid) for 2.5 min then hold at 95%Solvent B in Solvent A for 1 min (purity and low resolution MS only).

Unless otherwise noted, proton nuclear magnetic resonance (NMR) spectrawere obtained on either: (1) Bruker BBFO ASCEND™400 AVANCE IIIspectrometer at 400 MHz or (2) Bruker BBFO ULTRASHIELD™300 AVANCE IIIspectrometer at 300 MHz. Spectra are given in ppm (δ) and couplingconstants, J, are reported in Hertz (Hz). Tetramethylsilane (TMS) wasused as an internal standard. Mass spectra were collected using a WatersZQ Single Quad Mass Spectrometer (ion trap electrospray ionization(ESI)).

Example 1 HDAC Inhibition Assays

This example describes in vitro inhibition properties of exemplaryHDAC11 inhibitors for various HDACs. HDAC inhibition assays wereperformed using an electrophoretic mobility shift assay at Nanosyn, Inc.(Santa Clara, Calif.). Full length human recombinant HDAC proteins wereexpressed in the baculoviral system and purified by affinitychromatography. A human recombinant HDAC3 was co-expressed with nuclearreceptor corepressor (Ncor2). The following peptide substrates wereused: FAM-RHKK(Ac)-NH2 for HDAC3, HDAC6 and HDAC8; FITC-H3K27(Ac)-NH2for HDAC1, HDAC2 and HDAC10; FAM-RHKK(tri-fluor-Ac)-NH2 for HDAC4,HDAC5, HDAC7, HDAC9 and HDAC11. Reactions consisting of compound,enzyme, and substrate were performed in reaction buffer (comprised of100 mM HEPES, pH7.5, 25 mM KCl, 0.1% bovine serum albumin, 0.01% TritonX-100) at 25° C. and quenched by the addition of termination buffer (100mM HEPES, pH7.5, 0.01% Triton X-100, 0.05% SDS). The fluorescenceintensity of the electrophoretically separated de-acetylated product andsubstrate peptide were measured and analyzed using the LabChip® 3000microfluidic electrophoresis instrument (Perkin Elmer/Caliper LifeSciences). The IC₅₀ values of inhibitors were determined by fitting the%-inhibition curves with 4 parameter dose-response model using XLfit 4software (IDB S).

Table 1 shows the activity of exemplary selective HDAC11 inhibitors fromthree different chemical series.

Exemplary selective HDAC11 inhibitors HDTK010 and HDTK054display>200-fold selectivity over HDACs 1-10.

Exemplary selective HDAC11 inhibitors HDTK070 and HDTK028display>1800-fold selectivity over HDACs1-10.

TABLE 1 Chemical Compound HDAC Biochemical IC50 (μM) Series ID HDAC1HDAC2 HDAC3 HDAC4 HDAC5 HDAC6 HDAC7 HDAC8 HDAC9 HDAC10 HDAC11 AHDTK010 >10 >10 >10 >10 >10 0.5-1.0 5-10 0.5-1.0 >10 >10 0.001-0.005 AHDTK069 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 (inactive) BHDTK070 >10 >10 >10 >10 >10 >10 >10 5-10 >10 >10 0.001-0.005 BHDTK028 >10 >10 >10 >10 >10 >10 >10 10 >10 >10 0.001-0.005 BHDTK072 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 (inactive) CHDTK054 >10 >10 >10 >10 >10 >10 >10 5-10 >10 >10 0.01-0.05

Example 2 Bone Marrow Transplant Assay

This example describes certain in vivo effects of modulating HDAC11 invivo, using an exemplary model mouse for MPN disease. Effects of HDAC11knockout on MPN disease were determined using a MPLW515L mouse bonemarrow transplant model. This model was established using procedures asdescribed previously with minor modifications (Pikman et al. PloS Med.2006). Bone marrow cells were harvested from C57BL/6 donor mice (eitherwild-type (WT) or HDAC11 knock out (KO) mice) 7 days after5-fluorouracil injection (150 mg/kg). Cells were then treated with redblood cell lysis buffer and cultured overnight in transplantation medium(RPMI-1640+10% fetal bovine serum (FBS)+6 ng/ml IL-3, 10 ng/ml IL-6 and10 ng/ml stem-cell factor (SCF)) at 37° C. and 5% CO2. The next daycells were transduced with recombinant retroviruses overexpressingeither MPLW515L or MPLWT by spin infection at 2500 rpm for 90 minutes at30° C. The spin infection was repeated 24 hours later. Cells were thenre-suspended in PBS and injected into tail veins of lethally irradiated(2×450 cGy) C57BL/6 recipient mice at 0.8-1.0×106 cells/mouse. Viralconstructs used included MSCV-human-MPLW515L-green fluorescent protein(GFP) and MSCV-human-MPLWT-GFP. Peripheral blood cell counts includingwhite blood cells (WBC), platelets (PLT), and red blood cells (RBC) weremeasured at indicated time points to evaluate disease burden. Spleenswere isolated and the weights were measured. Bone marrow cells werecollected from two femurs and two tibias. After RBC lysis, cells werelysed for western blot analysis. Cells were lysed in RIPA buffer(Sigma-Aldrich, St. Louis, Mo.) supplemented with protease inhibitors(Roche, Basel, Switzerland) and phosphatase inhibitors (Sigma P5726).Protein concentrations were determined with Pierce BCA protein assay kit(Thermo Fisher Scientific, Waltham, Mass.). Proteins were separated on a4-12% Bis-Tris gradient electrophoresis gel (Life Technologies,Carlsbad, Calif.) and transferred onto a nitrocellulose membrane. Themembrane was then blocked in either 5% nonfat dry milk or 5% bovineserum albumin (BSA) followed by incubation with primary antibodies at 4°C. overnight. Antibodies for p-STAT5 (Abcam 32364), STAT5 (CellSignaling 9358), p-STAT3 (Cell Signaling 9145) and STAT3 (Cell Signaling9139) were used. β-actin was used as loading control.

The recipients of the MPL W515L transduced-WT bone marrow displayedsplenomegaly and increased platelet and red blood cell count (FIG. 1)compared to MPL WT transduced-WT bone marrow recipients. The recipientsof the MPL W515L transduced-HDAC11 KO bone marrow showed amelioration ofthe disease, with significantly fewer platelets and red blood cells andsmaller spleen size in comparison to the recipients of the MPL W515Ltransduced-WT bone marrow. These results demonstrated that HDAC11knockout reduces thrombocytosis and erythrocytosis in a MPN mouse model.

The amounts of STAT3 and STAT5 phosphorylation were examined by westernblots of whole bone marrow samples on Day 34. With WT donor bone marrowcells, transduction of MPL W515L resulted in increased STAT3 and STAT5phosphorylation, compared to transduction of MPL WT (FIG. 2).Transduction of MPL W515L in the HDAC11 KO donor bone marrow cellsresulted in inhibition of STAT3 and STAT5 phosphorylation.

Example 3 Cell Proliferation Assays in Cells Expressing MPL or JAK2Mutations

This example describes inhibition of in vitro cell proliferation withexemplary HDAC11 inhibitors. The effects of selective HDAC11 inhibitorson cell proliferation were measured in cell lines containing theMPN-associated mutations MPL W515L (Ba/F3 MPL W515L) and JAK2 V617F(Ba/F3 JAK2 V617F, HEL 92.1.7, SET-2). HEL 92.1.7 and SET-2 cells werepurchased from the ATCC and DSMZ, respectively. Ba/F3 cellsoverexpressing either MPLW515L or JAK2V617F, which were cytokineindependent, were generated by transforming Ba/F3 cells withretroviruses overexpressing either MPL W515L or JAK2V617F. For survivalassays, cells were seeded in 96-well plates at the density of 0.1million/ml with addition of indicated compounds and incubated at 37° C.for 48 h. CCK8 solution (Dojindo, Rockville, Md.) was added andabsorbance at 450 nm was measured after 3 hours of incubation. Readingswere normalized to dimethylsulfoxide (DMSO)-treated wells.

Exemplary selective HDAC11 inhibitors HDTK028 and HDTK054 inhibitedproliferation of Ba/F3 MPLW515L and Ba/F3 JAK2V617F with IC50 values inthe 1-10 μM range. The exemplary selective inhibitors HDTK010 andHDTK070 inhibited the growth of the HEL 92.1.7 cell line, while aninactive analog HDTK069 did not (FIG. 3). While less potent than theJAK2 inhibitor Ruxolitinib, the exemplary selective HDAC11 inhibitorsHDTK010 and HDTK070 inhibited the growth of a SET-2 cell line that wasgenerated to be Ruxolitinib-resistant (FIG. 4).

Example 4 Cell Cycle Assay

This example describes in vitro cell cycle inhibition mediated by anexemplary HDAC11 inhibitor.

The effect of selective HDAC11 compounds on cell cycle was determinedusing flow cytometry. After treatment, HEL 92.1.7 cells were fixed in70% ethanol overnight. The next day, cells were washed with PBS andsuspended in 4′,6-diamidino-2-phenylindole (DAPI)/Triton X-100 stainingsolution (0.1% Triton X-100, 1 ug/ml DAPI) and kept in dark for 1 hourbefore measured by a LSRII cytometer (BD Biosciences, San Diego,Calif.). Analysis was performed using ModFit LT software v4.1 (VeritySoftware House).

The exemplary selective HDAC11 inhibitor HDTK070 induced G1 cell cyclearrest in HEL 92.1.7 cells after 12 hours of treatment at 10 μMconcentration (FIG. 5).

Example 5 STAT3 and STAT5 Pathway Assays

The example describes in vitro cell expression and phosphorylation ofcertain JAK/STAT pathway polypeptides upon treatment with exemplaryHDAC11 inhibitors.

STAT3 and STAT5 Phosphorylation Western Blot Assay

The effects of selective HDAC11 inhibition on STAT3 and STAT3phosphorylation in HEL 92.1.7 cells were determined by western blot.Cells were treated with HDAC11 inhibitors and lysed in RIPA buffer(Sigma-Aldrich, St. Louis, Mo.) supplemented with protease inhibitors(Roche, Basel, Switzerland) and phosphatase inhibitors (Sigma P5726).Protein concentrations were determined with Pierce BCA protein assay kit(Thermo Fisher Scientific, Waltham, Mass.). Proteins were separated on a4-12% Bis-Tris gradient electrophoresis gel (Life Technologies,Carlsbad, Calif.) and transferred onto a nitrocellulose membrane. Themembrane was then blocked in either 5% nonfat dry milk or 5% BSAfollowed by incubation with primary antibodies at 4° C. overnight.Antibodies for p-STAT5 (Abcam 32364), STAT5 (Cell Signaling 9358),p-STAT3 (Cell Signaling 9145) and STAT3 (Cell Signaling 9139) were used.β—actin was used as loading control.

As shown in FIG. 6, the exemplary selective HDAC11 inhibitor HDTK010inhibited STAT3 and STAT5 phosphorylation.

JAK/STAT Signaling Pathway PCR Array

The effects of HDAC11 inhibition on the STAT pathway were furtherexamined using a JAK/STAT Signaling Pathway PCR Array (Qiagen). HEL92.1.7 cells were treated with HDTK010 for 24 or 48 hours. At the end oftreatment, the cells were lysed and total RNA was isolated using theRNeasy Plus Micro Kit following the manufacturer's protocol (Qiagen).RNA (0.5 μg) was converted to cDNA using the iScript reaction mix(BioRad). The cDNA template was mixed with RT2 SYBR Green Mastermix andadded to the PCR Array. Reactions were carried out in an EppendorfRealplex2 ep.gradient Mastercycler and data analyzed per manufacturer'sprotocol. Results from HDAC11 treatment were compared to DMSO control.

Table 2 lists genes in the JAK/STAT Signaling Pathway with increasedexpression after HDAC11 inhibitor treatment.

TABLE 2 JAK/STAT Signaling Pathway Genes Modulated by HDAC11 InhibitorPDGFR JUN IL10RA CDKN1A

Example 6

Colony Formation Assay from Patient Samples

To determine the effects of selective HDAC11 inhibition on MPN patientsamples, the effects of HDAC11 inhibitors were evaluated in a colonyformation assay with erythroid or myeloid cells from MPN patients.Peripheral blood was obtained from MPN patients consented through theMoffitt Cancer Center Total Cancer Care protocol (MCC 14690). Blood wastreated with HetaSep™ (STEMCELL Technologies, Inc.) to remove themajority of red blood cells. Peripheral blood mononuclear cells (PBMCs)were isolated by ficoll separation. PBMCs (1-4×105) were then plated in1 mL of methylcellulose medium containing rhSCF, rhIL-3, andgranulocyte-macrophage colony-stimulating factor (rhGM-CSF) (MethoCult™#H4534; STEMCELL Technologies, Inc.). All drug treated samples contained0.1% DMSO as the final concentration. Cells were incubated at 37° C. and5% CO2 in a humidified incubator. Colonies were scored 12 to 14 daysafter plating.

The results from two patient samples are shown in FIG. 7. One patienthad essential thrombocytosis (ET), and the other had polycythemia vera(PV) and was also Ruxolitinib resistant. The exemplary selective HDAC11inhibitor HDTK010 reduced the growth of colonies from both erythroid andmyeloid cells.

Example 7

Sphere Formation Assay with Stem-Like Side Population NSCLC Cells

In order to investigate the role of HDAC11 in the self-renewal ofstem-like cancer cells, sphere formation assays were conducted withNSCLC side population (SP) stem-like cells. A549 and NCI-H1650 cellswere obtained from the ATCC. Asynchronously growing A549 or NCI-H1650cells were washed once with phosphate buffered saline (PBS) andharvested using Accutase solution (Sigma Aldrich) and resuspended inDMEM-F12K medium (Gibo, Life Technologies) with 2% fetal bovine serum(FBS) at 1×106 cells/ml density. The cells were incubated with 4 μg/mlof Hoechst 33342 dye (Life Technologies) for 90 min at 37° C. in thepresence or absence of 1 M Fumitremorgin C (Sigma-Aldrich) which wasused as a control sample to set the gate during sorting. The Hoechststained cells were sorted using the BD FACSAria cell sorter. The sortedSP cells were plated in 96-well ultra-low adherent plate (Corning Inc)at the cell density of 1000 cells/100 μl/well in stem cell selectivemedium (DMEM:F12K supplemented with N2 supplement, 10 ng/mL epidermalgrowth factor (EGF) and 10 ng/ml basic fibroblast growth factor (bFGF)(Sigma-Aldrich) at 37° C. for 7-10 days. The spheres were observed andacquired using EVOS FL microscope system (Life Technologies Inc., USA).The numbers of spheres greater than or equal to 50 μm were counted. Tostudy the effect of the HDAC11 inhibitors, the appropriateconcentrations of the compounds were added to the wells on Day 0 and Day5 and the image acquisition and analysis of the spheres were performedon Day 10. The sphere formation assays were performed twice withtriplicates of each treatment in every assay.

As shown in FIG. 8, the exemplary selective HDAC11 inhibitors HDTK010,HDTK054, and HDTK070 prevented growth of the stem-like cancer cellscarrying either KRAS (A549) or EGFR (H1650) mutation, while the inactiveanalog HDTK072 did not.

Example 8

Tube Formation Assay with Stem-Like Side Population NSCLC Cells

The angiogenic tubule assay was performed with the sorted sidepopulation cells to analyze vascular mimicry. The H1650 side populationcells were sorted as mentioned above and they were further grown onMatrigel (BD Biosciences) to form angiogenic tubule-like structures. 100μl of thawed Matrigel was layered on the wells of 96-well tissue cultureplates and allowed to polymerize at 37° C. for 30 min. Sorted H1650 SPcells were layered (12000 cells/100 μl of Matrigel) on the polymerizedmatrigel and incubated overnight at 37° C. Tubule formation was assessedunder bright field using EVOS FL microscope system and images wereacquired with EVOS software (Life Technologies Inc., USA). For thetreatment with HDAC11 inhibitors, appropriate concentrations of thecompounds were added to the cells when they were seeded on Matrigel forthe tubule formation assay.

The exemplary selective HDAC11 inhibitors HDTK054 and HDTK070 inhibitedtubule formation by the H1650 SP cells, while the inactive analogHDTK072 did not (FIG. 9).

Example 9 Cell Migration Assay

The effects of HDAC11 inhibition on cell migration were determined usinga scratch assay. A549 cells (120,000-150,000 cells/2 ml/well) were grownin 6-well tissue culture plates (BD Biosciences). A scratch was madeusing a sterile 2 μl pipette tip in each well. The 10% FBS containingmedium was added to the wells after a wash with DPBS. To assess theeffect of the HDAC11 inhibitors on cell migration, the inhibitors wereadded in the medium at appropriate concentrations. The cells werefurther incubated at 37° C. The images were taken on Day 0 and afterevery 24 hours using EVOS FL microscope system and EVOS software (LifeTechnologies Inc) and the cell migration was compared to the Day 0 oftreatment. The exemplary selective HDAC11 inhibitors HDTK054 and HDTK070impaired A549 cell migration, while the effects of the inactive analogHTK072 were similar to the control (FIG. 10).

Example 10 QRT-PCR Assay for Stem Cell Transcription Factors

Given the effects of HDAC11 inhibition on stem-like side populationcells, the effects on the expression of stem cell transcription factorswas determined using QRT-PCR. A549 or H1650 cells were plated in 60 mmcell culture dishes (120,000 cells). The next day, HDAC11 inhibitorswere added at various concentrations and incubated for 72 hours. For thesiRNA experiments, A549 cells were plated in 60 mm cell culture dishes(120,000 cells per dish). The siRNAs (Santa Cruz Biotechnologiessc-106896 or ThermoFisher Scientific Cat#4392420) were transfected at aconcentration of 100 and 150 pmoles into cells using Oligofectaminereagent (Invitrogen) as per manufacturer's protocol. A non-targetingsiRNA (Ambion AM4635) was used as a control for all the transfectionexperiments. The cells were harvested after 72 hours post transfection.At the end of the treatments, cells were scraped, lysed and total RNAwas isolated by RNeasy miniprep kit from Qiagen following themanufacturer's protocol (RNeasy mini kit Cat No. 74104). 1 μg of RNA wasconverted into cDNA using iScript cDNA synthesis kit (BioRad) in a 20 μltotal reaction volume. Levels of mRNA were further analyzed usingquantitative real time-PCR (qRT-PCR) that was performed in CFX96 Realtime system using BioRad iQ SYBR Green supermix. The reaction was setwith 5 μl of SYBR Green, 1 μl of 1 μM Forward primer, 1 μl of 1 μMReverse primer, 0.5 μl of cDNA and 4.5 μl of water in a total 12 μlreaction. Data was normalized using GAPDH as an internal control andfold change was calculated by 2^(−Delta-Delta)Ct method. First theDelta-Ct was calculated (Delta-Ct=Ct Gene−Ct GAPDH). Next Delta-Delta-Ctwas calculated (Delta-Delta-Ct=Delta-Ct treatment−Delta-Ct control).Finally fold change was calculated by 2^(−Delta-Delta)Ct.

The primers used for amplification were as follows:

YAP1 FP (SEQ ID NO: 1) 5′-CCCAAGACGGCCAACGTGCC-3′, YAP1 RP(SEQ ID NO: 2) 5′-ACTGGCCTGTCGGGAGTGGG-3′,  Annealing Tm-58° C. Sox2 FP(SEQ ID NO: 3) 5′-GGGAAATGGGAGGGGTGCAAAAGA-3′, Sox2 RP (SEQ ID NO: 4)5′-TTGCGTGAGTGTGGATGGGATTGG-3′, Annealing Tm-55° C. Oct4 FP(SEQ ID NO: 5) 5′-ACATCAAAGCTCTGCAGAAAGAACT-3′, Oct4 RP (SEQ ID NO: 6)5′-CTGAATACCTTCCCAAATAGAACCC-3′, Annealing Tm-52° C. Nanog FP(SEQ ID NO: 7) 5′-AGAAGGCCTCAGCACCTA-3′, Nanog RP (SEQ ID NO: 8)5′-GGCCTGATTGTTCCAGGATT-3′, Annealing Tm-52° C. GAPDH FP (SEQ ID NO: 9)5′-GGTGGTCTCCTCTGACTTCAACA-3′, GAPDH RP (SEQ ID NO: 10)5′-GTTGCTGTAGCCAAATTCGTTGT-3′ Annealing Tm-52-60° C. Gli1 FP(SEQ ID NO: 11) 5′-CCCAATCACAAGTCAGGTTCCT-3′, Gli1 RP (SEQ ID NO: 12)5′-CCTATGTGAAGCCCTATTTGCC-3′ Annealing Tm-57° C.

HDAC11 and HDAC1 knockdown in A549 cells resulted in decreasedexpression of Sox2. The expression of two other stem cell transcriptionfactors, Oct4 and Nanog, was unchanged with HDAC1, HDAC6, or HDAC11knockdown (FIG. 11).

Treatment with exemplary selective HDAC11 inhibitors HDTK010, HDTK028,HDTK054, or HDTK070 also resulted in decreased expression of Sox2 inA549 and H1650 cells. In addition, decreased expression of Gli1 and Yap1was also observed (FIG. 12).

Example 11

Co-Culture Assay with Cancer Cells and Cancer Associated Fibroblasts

The effects of selective HDAC11 inhibition on cancer cells and cancerassociated fibroblasts (CAFs) were determined in a co-culture assay. Thecells were first trypsinized and collected in serum containing mediumand were further incubated in same media at 37° C. for 15-20 min forrecovery. The cells were resuspended in serum free media at 1×106cells/ml density and stained with 10 uM of cytotracker green (Cancerassociated fibroblasts) or cytotracker red dye (H1650 cells) (LifeTechnologies Inc) at 37° C. for 90 min. The stained cells were washedtwice with serum free medium to remove excess dye at 1500 rpm for 5 min.The cytotracker green stained CAFs were mixed with cytotracker redstained H1650 cells in 1:1 ratio and grown in 96-well tissue cultureplate overnight. The HDAC11 inhibitors were then added to the mixedadhered cells (CAFs+H1650) at appropriate concentrations. The imageswere taken on Day 0 and after every 24 hours using EVOS FL microscopesystem and EVOS software (Life Technologies Inc).

As shown in FIG. 13, the exemplary selective HDAC11 compounds HDTK054and HDTK070 inhibit the growth of H1650 cells co-cultured with primaryCAFs from human lung cancer with minimal effects on CAFs.

Example 12

Cell Proliferation Assays with Combination Treatment

Selective HDAC11 inhibitors may be useful in combination with othertargeted agents, including Jak2 inhibitors and Smoothened inhibitors.The effect of combination with the Smoothened inhibitors GDC-0449(Vismodegib) and BMS-833923 was tested in lung cancer cell lines. A549and NCI-H2170 cells were obtained from the ATCC. H2170 is a squamouscarcinoma cell line. Cell viability was assessed using MTT reagent(Thiazolyl Blue Tetrazolium Bromide) (Sigma-Aldrich). The cells weregrown in 96-well plates at a density of 3000-5000 cells/well intriplicates. The HDAC11 inhibitors were added to the adherent cells atdifferent concentrations and treatment was carried out for 96 hours.After treatments, the cells were incubated with the 1 mg/mL MTT reagentat 37° C. for 1 hour. The reaction was terminated with DMSO thatsolubilizes the formazan product formed. Absorbance at 590 nm wasrecorded in a plate reader.

Single agent treatment with the Smoothened inhibitors or exemplaryselective HDAC11 inhibitors had minimal effects on cell growth. However,the combination of exemplary selective HDAC11 inhibitors HDTK010,HDTK028, HDTK054 or HDTK070 with the Smoothened inhibitors resulted indecreased cell viability (FIG. 14)

Synthesis of Aminobenzimidazoles and Related Compounds Example 1-1.3-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-N-hydroxybenzamide

Step 1. methyl 3-(5,6-dichlorobenzo[d]oxazol-2-ylamino)benzoate

A solution of 2-amino-4,5-dichlorophenol (209 mg, 1.17 mmol) and methyl3-isothiocyanatobenzoate (226 mg, 1.17 mmol) in THF (17.5 mL) stirredfor 3 h at room temperature. Copper (II) sulfate (1.68 g, 11.5 mmol),triethyl amine (0.16 mL, 1.17 mmol), and silica gel (1.68 g) were added,and the resulting mixture stirred overnight at room temperature. Themixture was filtered, and the filtrate was concentrated. The residue waspurified vis column chromatography on silica gel (eluting with 10:1,dichloromethane/methanol) to afford methyl3-(5,6-dichlorobenzo[d]oxazol-2-ylamino)benzoate (100 mg, 25%) as awhite solid. MS: (ESI, m/z): 337[M+H]⁺.

Step 2. 3-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.55 mL, 8.9 mmol) and 1 Maqueous sodium hydroxide solution (0.60 mL, 0.60 mmol) were added to a0° C. solution of methyl3-(5,6-dichlorobenzo[d]oxazol-2-ylamino)benzoate (100 mg, 0.30 mmol) inTHF/MeOH (4:1, 5 mL), and the resulting solution stirred for 2 h at roomtemperature. The solution was cooled to 0° C., and pH of the mixture wasadjusted to 6 with 6 M aqueous HCl solution. The crude product waspurified by Prep-HPLC with the following conditions: Column, XBridgePrep C18, 5 um, 19×150 mm; Mobile phase A: water with 0.05% TFA; Mobilephase B: ACN; Gradient: 5%-95% B in 2 min; Detector, 254 nm. Thecollected fraction was lyophilized to give3-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-N-hydroxybenzamide (3.6 mg, 3%)as an off-white solid. ¹H-NMR (DMSO, 300 MHz), δ (ppm): 11.22 (s, 1H),11.05 (s, 1H), 9.06 (s, 1H), 8.08 (s, 1H), 7.95-7.89 (m, 2H), 7.77 (s,1H), 7.49-7.38 (m, 2H). MS: (ESI, m/z): 338[M+H]⁺.

Example 2-1.3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)-N-hydroxybenzamide

Step 1. 5-bromo-2-nitro-4-(trifluoromethyl)phenol

Potassium tert-butoxide (1.68 g, 14.97 mmol) was added in portions to a−60° C. solution of liquid ammonia (20 mL). A solution of tert-butylhydroperoxide (590 mg, 6.55 mmol) and1-bromo-4-nitro-2-(trifluoromethyl)benzene (1.61 g, 5.96 mmol) intetrahydrofuran (6 mL) was added, and the resulting solution was stirredfor 30 min at −30° C. The reaction was then quenched by the addition of50 mL of saturated aqueous ammonium chloride, and the resulting mixturewas extracted with 3×50 mL of ethyl acetate. The combined organic layerswere concentrated under vacuum. The crude product was purified byrecrystallization from petroleum ether/ethyl acetate (10:1) to give5-bromo-2-nitro-4-(trifluoromethyl)phenol (1.5 g, 88%) as a yellowsolid. MS: (ESI, m/z): 284[M−H]⁻.

Step 2. 2-amino-5-bromo-4-(trifluoromethyl)phenol

Hydrogen gas was introduced into a mixture of5-bromo-2-nitro-4-(trifluoromethyl)phenol (500 mg, 1.75 mmol) and Raneynickel (50 mg) in methanol (10 mL), and the reaction mixture stirred for2 h at room temperature. The solids were filtered out, and the filtratewas concentrated under vacuum to give2-amino-5-bromo-4-(trifluoromethyl)phenol (430 mg, 96%) as a blacksolid. MS: (ESI, m/z): 256[M+H]⁺.

Step 3. methyl3-(6-bromo-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate

A mixture of 2-amino-5-bromo-4-(trifluoromethyl)phenol (330 mg, 1.29mmol), methyl 3-isothiocyanatobenzoate (300 mg, 1.55 mmol),triethylamine (0.22 mL, 1.59 mmol), copper (II) sulfate (2.2 g), andsilica gel (2.8 g) in tetrahydrofuran (30 mL) stirred for 3 h at 50° C.The reaction mixture was filtered, and the filtrate was concentratedunder vacuum. The residue was purified via column chromatography onsilica gel (eluting with 50% ethyl acetate/petroleum ether) to affordmethyl 3-(6-bromo-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate(260 mg, 49%) as a yellow solid. MS: (ESI, m/z): 415[M+H]⁺.

Step 4. methyl3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate

A solution of methyl3-(6-bromo-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate (87 mg,0.21 mmol), zinc cyanide (23 mg, 0.20 mmol), DavePhos (33 mg, 0.08mmol), Pd₂(dba)₃ (60 mg, 0.05 mmol) in DMA (5 mL) stirred for 1 h at 90°C. in an oil bath. The resulting mixture was cooled to room temperatureand then diluted 30 mL of water. The resulting solution was extractedwith 3×50 mL of ethyl acetate, and the combined organic phases werewashed with 100 mL of brine, dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum. The residue was purified viacolumn chromatography on silica gel (gradient elution with 20-50% ethylacetate/petroleum ether) to give methyl3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate (65 mg,86%) as a light yellow solid. MS: (ESI, m/z): 362[M+H]⁺.

Step 5. 3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoicacid

A solution of methyl3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate (145 mg,0.40 mmol) 1 M aqueous lithium hydroxide solution (2 mL, 2 mmol) intetrahydrofuran (2 mL) stirred overnight at room temperature. The pHvalue of the reaction mixture was adjusted to 5 with 2 M aqueous HClsolution. The resulting solution was diluted with 10 mL of water andextracted with 3×10 mL of ethyl acetate. The combined organic phaseswere washed with 30 mL of brine, dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum. The residue was purified byreversed phase column with the following conditions: Column: C18 column,40 g, 20-45 um, 100 A; Mobile phase: water with 0.05% TFA and ACN (20%up to 50% in 30 minutes), 254 & 220 nm. The collected fraction wasconcentrated under vacuum to give3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoic acid (103mg, 74%) as a light yellow solid. MS: (ESI, m/z): 348[M+H]⁺.

Step 6.3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)-N-hydroxybenzamide

A solution of3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoic acid (103mg, 0.30 mmol), NMM (149 mg, 1.48 mmol), hydroxylamine hydrochloride (22mg, 0.33 mmol), IPCF (0.041 mL, 0.30 mmol) in DMA (3 mL) stirred for 4 hat room temperature. The crude product was purified by Prep-HPLC withthe following conditions: Column: Waters HSS C18, 2.1×50 mm, 1.8 um;Mobile Phase A:water/0.05% TFA; Mobile Phase B: ACN/0.05% TFA; Flowrate: 0.7 mL/min; Gradient: 5% B to 95% B in 2.0 min, hold 0.6 min; UV254 nm. The collected fraction was lyophilized to give3-(6-cyano-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)-N-hydroxybenzamide(34.9 mg, 32%) as an off-white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.44 (s, 1H), 11.24 (s, 1H), 9.07 (s, 1H), 8.45 (s, 1H), 8.09 (d,J=10.8 Hz, 2H), 7.93-7.91 (m, 1H), 7.51-7.43 (m, 2H). MS: (ESI, m/z):363[M+H]⁺.

Example 3-1.N-hydroxy-3-(6-phenyl-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzamide

Step 1. methyl3-(6-phenyl-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate

A solution of methyl3-(6-bromo-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate (80 mg,0.19 mmol), Pd(PPh₃)₄(45 mg, 0.04 mmol), sodium carbonate (41 mg, 0.39mmol) and phenylboronic acid (26 mg, 0.21 mmol) in 1,4-dioxane (5 mL)stirred for 2 h at 100° C. in an oil bath. The resulting mixture wascooled to room temperature and concentrated under vacuum. The residuewas purified via column chromatography on silica gel (gradient elutionwith 10-50% ethyl acetate/petroleum ether) to give methyl3-(6-phenyl-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate (50 mg,63%) as a yellow solid. MS: (ESI, m/z): 413 [M+H]⁺.

Step 2:N-hydroxy-3-(6-phenyl-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzamide

Hydroxyl amine solution (50% in water, 0.37 mL, 6.1 mmol) and 1 Maqueous sodium hydroxide solution (0.20 mL, 0.20 mmol) were added to asolution of methyl3-(6-phenyl-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzoate (40 mg,0.10 mmol) in THF/MeOH (4:1, 2 mL), and the resulting solution stirredovernight at room temperature. The crude product was purified byprep-HPLC with the following conditions: Column: XBridge RP C18, 19×150mm, 5 um; Mobile Phase A:water/0.05% TFA, Mobile Phase B: ACN; Flowrate: 25 mL/min; Gradient:5% B to 50% B in 6.0 min, 254 nm. Thecollected fraction was lyophilized to giveN-hydroxy-3-(6-phenyl-5-(trifluoromethyl)benzo[d]oxazol-2-ylamino)benzamide(11.4 mg, 28%) as off-white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.23 (s, 1H), 11.10 (s, 1H), 8.13 (s, 1H), 7.97-7.91 (m, 2H), 7.58 (s,1H), 7.50-7.36 (m, 7H). MS: (ESI, m/z): 414[M+H]⁺.

Example 4-1.5-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-2-fluoro-N-hydroxybenzamide

Intermediate 1. 5,6-dichlorobenzo[d]oxazol-2-amine

A solution of 2-amino-4,5-dichlorophenol (500 mg, 2.81 mmol), cyanogenbromide (356 mg, 3.36 mmol) in methanol (5 mL) stirred for 6 h at roomtemperature. The reaction was then poured into 50 mL of 2 M aqueoussodium bicarbonate solution and extracted with 3×20 mL of ethyl acetate.The combined organic phases were washed with 50 mL of brine, dried overanhydrous sodium sulfate, filtered, and concentrated under vacuum. Theresidue was purified vis column chromatography on silica gel (elutingwith 25% ethyl acetate-petroleum ether) to afford5,6-dichlorobenzo[d]oxazol-2-amine (340 mg, 60%) as a yellow solid. MS:(ESI, m/z): 203 [M+H]⁺.

Step 1. methyl 5-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-2-fluorobenzoate

A solution of t-BuBrettPhos (24 mg, 0.049 mmol) and Pd(OAc)₂ (5.6 mg,0.02 mmol) in water (0.05 mL) and tert-butanol (2 mL) was added to a10-mL sealed tube, and the system was purged and maintained with aninert atmosphere of nitrogen. The resulting solution stirred for 2 minat 110° C. and was then transferred via cannula over 1 minute to a 10-mLsealed tube charged with a solution of5,6-dichlorobenzo[d]oxazol-2-amine (100 mg, 0.49 mmol), methyl5-bromo-2-fluorobenzoate (138 mg, 0.59 mmol), and potassium carbonate(104 mg, 0.75 mmol) in tert-butanol (2 mL) under an inert atmosphere ofnitrogen. The resulting solution stirred overnight at 110° C. Thereaction mixture was cooled to room temperature and then diluted with 15mL of water. The resulting solution was extracted with 3×15 mL of ethylacetate, and the combined organic phases were dried over anhydroussodium sulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:10)) to give methyl5-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-2-fluorobenzoate (70 mg, 40%)as a yellow solid. MS: (ESI, m/z): 355[M+H]⁺.

Step 2.5-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-2-fluoro-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.73 mL, 12 mmol) and 1 M aqueoussodium hydroxide solution (0.40 mL, 0.40 mmol) were added to a solutionof methyl 5-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-2-fluorobenzoate (70mg, 0.20 mmol) in THF/MeOH (4:1, 3 mL), and the resulting solutionstirred for 2 h at room temperature. The solids were filtered out, andthe crude product was purified by prep-HPLC with the followingconditions: Column, Xbridge RP18 5 um, 19×150 mm; mobile phase, water(0.05% TFA) and CH₃CN (5% CH₃CN up to 35% in 7 min); Detector, UV220/254 nm. The collected fraction was lyophilized to give5-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-2-fluoro-N-hydroxybenzamide(11.7 mg, 17%) as an off-white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.12 (s, 1H), 11.03 (s, 1H), 9.27 (s, 1H), 8.00-7.91 (m, 2H), 7.83-7.72(m, 2H), 7.36-7.31 (m, 1H). MS: (ESI, m/z): 356[M+H]⁺.

The following compounds were prepared according to the proceduresoutlined above for5-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-2-fluoro-N-hydroxybenzamide.

(ESI, m/z) Ex. Structure Name ¹H NMR [M + H]⁺ 4-2

3-((5,6- dichlorobenzo[d] oxazol-2-yl) amino)-2-fluoro- N-hydroxy-benzamide (DMSO, 300 MHz, ppm): 11.07 (s, 1H), 10.84 (s, 1H), 9.27 (s,1H), 8.27-8.22 (m, 1H), 7.95 (s, 1H), 7.73 (s, 1H), 7.34-7.23 (m, 2H)356 4-3

3-((5,6- dichlorobenzo[d] oxazol-2-yl) amino)-4-fluoro- N-hydroxy-benzamide (DMSO, 300 MHz, ppm): 11.25 (s, 1H), 10.83 (s, 1H), 9.10 (s,1H), 8.51 (d, J = 7.5 Hz, 1H), 7.95 (s, 1H), 7.77 (s, 1H), 7.55- 7.50(m, 1H), 7.43-7.37 (m, 1H) 356 4-4

3-((5,6- dichlorobenzo[d] oxazol-2-yl) amino)-5-fluoro- N-hydroxy-benzamide (DMSO, 400 MHz, ppm): 11.31 (s, 1H), 9.18 (s, 1H), 8.19 (s,1H), 8.04 (d, J = 10.8 Hz, 1H), 7.98-7.89 (m, 1H), 7.82 (s, 2H), 7.20(d, J = 9.2 Hz, 1H). 356 4-5

5-((5,6- dichlorobenzo[d] oxazol-2-yl) amino)-N- hydroxy- nicotinamide(DMSO, 300 MHz, ppm): 10.96 (br, 1H), 9.23 (br, 1H), 8.93 (s, 1H),8.54-8.51 (m, 2H), 7.94 (s, 1H), 7.78 (s, 1H) 339 4-6

4-((5,6- dichlorobenzo[d] oxazol-2-yl) amino)-N- hydroxy- picolinamide(DMSO, 400 MHz, ppm): 11.57 (br, 1H), 11.39 (br, 1H), 9.08 (s, 1H), 8.50(d, J = 5.6 Hz, 1H), 8.27 (s, 1H), 8.03 (s, 1H), 7.88 (d, J = 3.2 Hz,2H) 339 4-7

2-((5,6- dichlorobenzo[d] oxazol-2-yl) amino)-N- hydroxyiso-nicotinamide (DMSO, 400 MHz, ppm): 11.75 (br, 1H), 11.57 (br, 1H), 9.31(s, 1H), 8.46-8.38 (m, 2H), 8.03 (s, 1H), 7.86 (s, 1H), 7.32 (d, J = 4.8Hz, 1H) 339 4-8

2-((5,6- dichlorobenzo[d] oxazol-2-yl) amino)-N- hydroxy- pyrimidine-4-carboxamide (DMSO, 400 MHz, ppm): 11.95 (br, 1H), 11.14 (br, 1H), 9.46(s, 1H), 8.89-8.84 (m, 1H), 8.03 (s, 1H), 7.86 (s, 1H), 7.53 (d, J = 4.4Hz, 1H) 340

Example 5-1.3-(5,6-dichlorobenzo[d]thiazol-2-ylamino)-N-hydroxybenzamide2,2,2-trifluoroacetate

Step 1: methyl 3-(5,6-dichlorobenzo[d]thiazol-2-ylamino)benzoate

A solution of 4,5-dichloro-2-iodoaniline (288 mg, 1 mmol), methyl3-isothiocyanatobenzoate (193 mg, 1 mmol), copper(I) bromide (14.2 mg,0.1 mmol), and TBAB (322 mg, 1 mmol) in DMSO (7 mL) stirred overnight at40° C. The reaction was cooled to room temperature and then poured intoof 30 mL of ice/water. The resulting solution was extracted with 2×50 mLof ethyl acetate, and the combined organic phases were washed with 20 mLof brine, dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:3)) to give methyl3-(5,6-dichlorobenzo[d]thiazol-2-ylamino)benzoate (238 mg, 67%) as anoff-white solid. MS: (ESI, m/z): 353[M+H]⁺.

Step 2: 3-(5,6-dichlorobenzo[d]thiazol-2-ylamino)-N-hydroxybenzamide2,2,2-trifluoroacetate

Hydroxyl amine solution (50% in water, 1.45 mL, 23.9 mmol) and 1 Maqueous sodium hydroxide solution (0.40 mL, 0.40 mmol) were added to asolution of methyl 3-(5,6-dichlorobenzo[d]thiazol-2-ylamino)benzoate (70mg, 0.20 mmol) in THF/MeOH (4:1, 8 mL), and the resulting solutionstirred for 2 h at room temperature. The solids were filtered out andthe crude product was purified by Prep-HPLC with the followingconditions: Column, Xbridge RP18 5 um, 19×150 mm; mobile phase, water(0.05% TFA) and CH₃CN (5% CH₃CN up to 85% in 8 min); Detector, UV220/254 nm. The collected fraction was lyophilized to give3-(5,6-dichlorobenzo[d]thiazol-2-ylamino)-N-hydroxybenzamide2,2,2-trifluoroacetate (17.7 mg, 19%) as a white solid. ¹H-NMR (DMSO,400 MHz), δ (ppm): 11.22 (s, 1H), 10.84 (s, 1H), 9.06 (s, 1H), 8.17 (s,1H), 8.02-8.00 (m, 2H), 7.83 (s, 1H), 7.48-7.37 (m, 2H). MS: (ESI, m/z):354[M+H]⁺.

Example 6-1.3-(5-cyano-6-(methylsulfonyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 5-(methylthio)-2-nitrobenzenamine

A solution of 5-chloro-2-nitroaniline (15.0 g, 86.7 mmol) and sodiumthiomethoxide (24.0 g, 346 mmol) in N,N-dimethylformamide (100 mL)stirred for 20 h at 65° C. in an oil bath. The reaction mixture wascooled to room temperature and diluted with 500 mL of ethyl acetate. Theresulting mixture was washed with 5×400 mL of water and 500 mL of brine,dried over anhydrous sodium sulfate, filtered, and concentrated undervacuum. The residue was purified via column chromatography with silicagel (eluting with ethyl acetate/petroleum ether (1:3)) to give5-(methylthio)-2-nitrobenzenamine (12 g, 75%) as a red solid. MS: (ESI,m/z): 185 [M+H]⁺.

Step 2: 4-bromo-5-(methylthio)-2-nitrobenzenamine

Bromine (9.87 mL, 30.8 g, 193 mmol) was added dropwise to a solution of5-(methylthio)-2-nitrobenzenamine (12 g, 65 mmol) in acetic acid (100mL), and the resulting solution stirred for 12 h at room temperature.The reaction mixture was then added dropwise into 500 mL of saturatedaqueous NaHSO₃ solution. The resulting mixture was extracted with 3×200mL of ethyl acetate, and the combined organic phases were washed with500 mL of brine, dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum to give4-bromo-5-(methylthio)-2-nitrobenzenamine (9.0 g, 53%) as a red solid.MS: (ESI, m/z): 263 [M+H]⁺.

Step 3: 4-amino-2-(methylthio)-5-nitrobenzonitrile

A solution of 4-bromo-5-(methylthio)-2-nitrobenzenamine (9.0 g, 34mmol), copper(I) cyanide (7.6 g, 85.4 mmol) and copper(I) iodide (810mg, 4.25 mmol) in DMF (80 mL) stirred for 12 h at 150° C. in an oilbath. The reaction mixture was cooled to room temperature and thenpoured into 1000 mL of water. The resulting mixture was extracted with3×500 mL of ethyl acetate, and the combined organic phases were washedwith 500 mL of brine, dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:3)) to give 4-amino-2-(methylthio)-5-nitrobenzonitrile (3.5 g,49%) as a yellow solid. MS: (ESI, m/z): 210[M+H]⁺.

Step 4: 4,5-diamino-2-(methylthio)benzonitrile

A solution of 4-amino-2-(methylthio)-5-nitrobenzonitrile (3.5 g, 16.7mmol) and tin (II) chloride dehydrate (18.8 g, 83.3 mmol) in ethylacetate (50 mL) and ethanol (25 mL) stirred for 2 h at 75° C. in an oilbath. The resulting solution was cooled to room temperature and thendiluted with 500 mL of ethyl acetate. The resulting mixture was washedwith 500 mL of 2 M aqueous sodium bicarbonate solution and 500 mL ofbrine, dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography withsilica gel (eluting with ethyl acetate/petroleum ether (1:1)) to give4,5-diamino-2-(methylthio)benzonitrile (1.5 g, 50%) as a yellow solid.MS: (ESI, m/z): 180[M+H]⁺.

Step 5: methyl3-(5-cyano-6-(methylthio)-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of 4,5-diamino-2-(methylthio)benzonitrile (1.00 g, 5.58mmol), methyl 3-isothiocyanatobenzoate (1.07 g, 5.54 mmol), EDC-HCl (3.2g, 16.7 mmol) in THF (10 mL) stirred for 3 h at 60° C. in an oil bath.The resulting solution was cooled to room temperature and then dilutedwith 100 mL of water. The resulting solution was extracted with 3×50 mLof ethyl acetate, and the combined organic phases were washed with 100mL of brine, dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:1)) to give methyl3-(5-cyano-6-(methylthio)-1H-benzo[d]imidazol-2-ylamino)benzoate (400mg, 21%) as a red solid. MS: (ESI, m/z): 339[M+H]⁺.

Step 6: methyl3-(5-cyano-6-(methylsulfonyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

MCPBA (610 mg, 3.53 mmol) was added portion wise to a solution of methyl3-(5-cyano-6-(methylthio)-1H-benzo[d]imidazol-2-ylamino)benzoate (400mg, 1.18 mmol) in dichloromethane (50 mL) and stirred for 12 h at roomtemperature. The reaction mixture was diluted with 100 mL ofdichloromethane, and the resulting solution was washed with 2×100 mL ofsaturated aqueous NaHSO₃ solution, 2×100 mL of saturated sodiumbicarbonate solution, and 100 mL of brine, dried over anhydrous sodiumsulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (7:10)) to give methyl3-(5-cyano-6-(methylsulfonyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(100 mg, 23%) as a red solid. MS: (ESI, m/z): 371 [M+H]⁺.

Step 7:3-(5-cyano-6-(methylsulfonyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.50 mL, 8.1 mmol) and 1 Maqueous sodium hydroxide solution (0.54 mL, 0.54 mmol) were added to asolution of methyl3-(5-cyano-6-(methylsulfonyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(100 mg, 0.27 mmol) in THF/MeOH (4:1, 2 mL), and the resulting solutionstirred for 1 h at room temperature. The crude product was purified byprep-HPLC with the following conditions: Column: XBridge RP C18, 19×150mm, 5 um; Mobile Phase A: water/0.05% TFA; Mobile Phase B: ACN; Flowrate: 25 mL/min; Gradient: 14% B to 14% B in 13.0 min, 254 nm. Thecollected fraction was lyophilized to give3-(5-cyano-6-(methylsulfonyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(11.7 mg, 12%) as a brown solid. ¹H-NMR (DMSO, 300 MHz), δ (ppm): 11.22(s, 1H), 10.35 (s, 1H), 8.05-7.99 (m, 4H), 7.47-7.35 (m, 2H), 3.35 (s,3H). MS: (ESI, m/z): 372[M+H]⁺.

Example 7-1.N-hydroxy-3-(6-(methylsulfonyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide

Step 1: N-(3-bromo-4-(trifluoromethyl)phenyl)acetamide

A solution of 3-bromo-4-(trifluoromethyl)aniline (10.0 g, 41.66 mmol) inacetic anhydride (50 mL) stirred for 12 h at room temperature, and wasthen concentrated under vacuum. The residue was recrystallized from 200mL of hexane to give N-(3-bromo-4-(trifluoromethyl)phenyl)acetamide(10.3 g, 87%) as a white solid. MS: (ESI, m/z): 282 [M+H]⁺.

Step 2: N-(5-bromo-2-nitro-4-(trifluoromethyl)phenyl)acetamide

N-(3-Bromo-4-(trifluoromethyl)phenyl)acetamide (6.00 g, 21.3 mmol) wasadded portionwise to sulfuric acid (24 mL), and the mixture was cooledto 0° C. Fuming nitric acid (3.1 mL, 10.7 mmol) was added dropwise at 0°C., and the resulting solution was stirred for 2 hours at 0° C. Thereaction mixture was added dropwise to 500 mL of ice/water, and theresulting mixture was filtered. The filter cake was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:100)) to giveN-(5-bromo-2-nitro-4-(trifluoromethyl)phenyl)acetamide (5.0 g, 72%) as alight yellow solid. MS: (ESI, m/z): 325[M−H]⁻.

Step 3: N-(5-(methylthio)-2-nitro-4-(trifluoromethyl)phenyl)acetamide

A solution of N-(5-bromo-2-nitro-4-(trifluoromethyl)phenyl)acetamide(3.000 g, 9.17 mmol) and sodium thiomethoxide (2.6 g, 18.5 mmol) inN,N-dimethylformamide (150 mL) stirred for 12 h at 70° C. in an oilbath. The resulting solution was cooled to room temperature and slowlypoured into 1000 mL of water. The solids were collected by filtrationand dried under vacuum to giveN-(5-(methylthio)-2-nitro-4-(trifluoromethyl)phenyl)acetamide (1.3 g,48%) as a yellow solid. MS: (ESI, m/z): 293[M−H]⁻.

Step 4: 5-(methylthio)-2-nitro-4-(trifluoromethyl)benzenamine

A solution ofN-(5-(methylthio)-2-nitro-4-(trifluoromethyl)phenyl)acetamide (700 mg,2.38 mmol) in 6 M aqueous HCl solution (100 mL) stirred for 2 h at 98°C. in an oil bath. The reaction was cooled to 0° C. and the pH value ofthe solution was adjusted to 8-9 with 6 M aqueous sodium hydroxidesolution. The resulting solution was extracted with 3×50 mL of ethylacetate, and the combined organic phases were washed with 1×100 mL ofbrine, dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography withsilica gel (eluting with ethyl acetate/petroleum ether (5:1)) to give5-(methylthio)-2-nitro-4-(trifluoromethyl)benzenamine (430 mg, 72%) as alight yellow solid. MS: (ESI, m/z): 251[M−H]⁻.

Step 5: 4-(methylthio)-5-(trifluoromethyl)benzene-1,2-diamine

A mixture of 5-(methylthio)-2-nitro-4-(trifluoromethyl)benzenamine (430mg, 1.70 mmol) and 10% palladium on carbon (50 mg) in methanol (100 mL)under an atmosphere of hydrogen gas stirred for 2 h at room temperature.The reaction mixture was filtered, and the filtrate was concentratedunder vacuum to give4-(methylthio)-5-(trifluoromethyl)benzene-1,2-diamine (330 mg, 87%) as adark red solid. MS: (ESI, m/z): 223[M+H]⁺.

Step 6: methyl3-(6-(methylthio)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of 4-(methylthio)-5-(trifluoromethyl)benzene-1,2-diamine (100mg, 0.45 mmol), and methyl 3-isothiocyanatobenzoate (86 mg, 0.45 mmol)in THF (10 mL) stirred for 4 h at 65° C. in an oil bath. EDC-HCl (260mg, 1.35 mmol) was added, and the resulting solution stirred for anadditional 1 h at 65° C. The reaction mixture was cooled to roomtemperature and the diluted with 30 mL of ethyl acetate. The resultingmixture was washed with 2×10 mL of brine, dried over anhydrous sodiumsulfate and concentrated under vacuum. The residue was purified viacolumn chromatography with silica gel (eluting withdichloromethane/methanol (10:1)) to give methyl3-(6-(methylthio)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(90 mg, 52%) as a yellow solid. MS: (ESI, m/z): 382[M+H]⁺.

Step 7: methyl3-(6-(methylsulfonyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of methyl3-(6-(methylthio)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(120 mg, 0.31 mmol) and MCPBA (162.8 mg, 0.94 mmol) in dichloromethane(10 mL) stirred for 12 h at room temperature. The reaction mixture wasdiluted with 20 mL of dichloromethane, and washed with 2 M aqueoussodium bisulfite solution (10 mL), 2 M aqueous sodium bicarbonatesolution (10 mL), and brine (10 mL), dried over anhydrous sodiumsulfate, filtered, and concentrated under vacuum to give methyl3-(6-(methylsulfonyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(50 mg, 38%) as a white solid. MS: (ESI, m/z): 414[M+H]⁺.

Step 8:N-hydroxy-3-(6-(methylsulfonyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide

Hydroxyl amine solution (50% in water, 0.22 mL, 3.6 mmol) and 1 Maqueous sodium hydroxide solution (0.24 mL, 0.24 mmol) were added to asolution of methyl3-(6-(methylsulfonyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(50 mg, 0.12 mmol) in THF/MeOH (4:1, 4 mL), and the resulting solutionstirred for 1 h at room temperature. The crude product was purified byprep-HPLC with the following conditions: Column: XBridge RP C18, 19×150mm, 5 um; Mobile Phase A: water/0.05% TFA, Mobile Phase B: ACN; Flowrate: 25 mL/min; Gradient: 5% B to 40% B in 7.0 min, 254 nm. Thecollected fraction was lyophilized to giveN-hydroxy-3-(6-(methylsulfonyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide(2.2 mg, 4%) as a brown solid. ¹H-NMR (DMSO, 300 MHz), δ (ppm): 11.24(br, 1H), 10.43 (br, 1H), 10.22 (br, 2H), 8.16 (s, 1H), 8.07 (s, 1H),7.99 (d, J=7.8 Hz, 1H), 7.91 (s, 1H), 7.47-7.35 (m, 2H), 3.26 (s, 3H).MS: (ESI, m/z): 415[M+H]⁺.

Example 8-1. 3-(1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: methyl 3-(1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of 2-bromo-1H-1,3-benzodiazole (50 mg, 0.25 mmol), methyl3-aminobenzoate (115 mg, 0.76 mmol), and 6 M aqueous HCl solution (1drop) in ethanol (3 mL) was irradiated with microwave radiation for 1 hat 120° C. The resulting mixture was cooled to room temperature and thenconcentrated under vacuum. The residue was purified by preparative thinlayer chromatography (eluting with dichloromethane/methanol (20:1)) toafford methyl 3-(1H-benzo[d]imidazol-2-ylamino)benzoate (78 mg, crude)as a yellow solid. MS: (ESI, m/z): 268[M+H]⁺.

Step 2: 3-(1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 1.1 mL, 17.5 mmol) and 1 Maqueous sodium hydroxide solution (0.58 mL, 0.58 mmol) were added to asolution of methyl 3-(1H-benzo[d]imidazol-2-ylamino)benzoate (78 mg,0.29 mmol) in THF/MeOH (4:1, 10 mL), and the resulting solution stirredfor 2 h at room temperature. The solids were filtered out, the crudeproduct was purified by prep-HPLC with the following conditions: Column:Waters HSS C18, 2.1×50 mm, 1.8 um; Mobile Phase A: water/0.05% TFA;Mobile Phase B: ACN/0.05% TFA; Flow rate: 0.7 mL/min; Gradient: 5% B to95% B in 2.0 min, hold 0.6 min; UV 254 nm. The collected fraction waslyophilized to give 3-(1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(53.2 mg, 68%) as a pink solid. ¹H-NMR (DMSO, 300 MHz), δ (ppm): 12.95(br, 1H), 11.28 (br, 1H), 10.94 (br, 1H), 9.13 (br, 1H), 7.87 (s, 1H),7.69-7.53 (m, 3H), 7.44-7.40 (m, 2H), 7.27-7.24 (m, 2H). MS: (ESI, m/z):269[M+H]⁺.

Example 9-1.3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)(methyl)amino)-N-hydroxybenzamide

Step 1:2-mercapto-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile

A solution of 4,5-diamino-2-(trifluoromethyl)benzonitrile (2.00 g, 9.94mmol), carbon disulfide (6.04 g, 79.52 mmoL) and potassium hydroxide(1.67 g, 29.8 mmol) in ethanol (100 mL) stirred for 5 h at 90° C. in anoil bath. The resulting mixture was cooled to room temperature andconcentrated under vacuum. The residue was diluted with 200 mL of water,and the resulting solution was extracted with 3×200 mL of ethyl acetate.The combined organic phases were washed with 100 mL of brine, dried overanhydrous sodium sulfate, filtered, and concentrated under vacuum togive 2-mercapto-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile(1.9 g, 79%) as a red solid. MS: (ESI, m/z): 244[M+H]⁺.

Step 2: 2-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile

Bromine (0.2 mL, 4.0 mmol) was added dropwise with stirring over 10 minto a 0° C. solution of2-mercapto-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile (267mg, 1.10 mmol) in hydrogen bromide (40% in acetic acid, 12 mL). Theresulting solution stirred for 3 h at 0° C. and was then diluted with 1mL of water. The pH value of the solution was adjusted to 4 with 1 Maqueous sodium hydroxide solution. The resulting solution was extractedwith 3×100 mL of ethyl acetate, and the combined organic phases weredried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum.The residue was purified via column chromatography with silica gel(eluting with ethyl acetate/petroleum ether (3:2)) to give2-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile (225 mg,71%) as a red solid. MS: (ESI, m/z): 290[M+H]⁺.

Step 3: methyl3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)(methyl)amino)benzoate

A solution of2-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile (130 mg,0.45 mmol), methyl 3-(methylamino)benzoate (101.9 mg, 0.62 mmol), and 12M aqueous HCl solution (1 drop) in ethanol (3 mL) was irradiated withmicrowave radiation for 1 h at 150° C. The resulting mixture wasconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (2:3)) to give methyl3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)(methyl)amino)benzoate(160 mg, 95%) as a reddish solid. MS: (ESI, m/z): 375[M+H]⁺.

Step 4:3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)(methyl)amino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.87 mL, 14.3 mmol) and 1 Maqueous sodium hydroxide solution (0.95 mL, 0.95 mmol) were added to asolution of methyl3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)(methyl)amino)benzoate(178 mg, 0.48 mmol) in THF/MeOH (4:1, 2 mL), and the resulting solutionstirred for 3 h at room temperature. The crude product was purified byprep-HPLC with the following conditions: Column, XSelect CSH Prep C18OBD Column, 19×150 mm 5 um 13 nm; mobile phase, Mobile Phase A:water/0.05% TFA, Mobile Phase B: ACN; Gradient: 15% B to 35% B in 7 min;Detector, 254 nm. The collected fraction was lyophilized to give3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)(methyl)amino)-N-hydroxybenzamide(45 mg, 25%) as a white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm): 11.29(s, 1H), 7.86-7.84 (m, 2H), 7.73-7.66 (m, 3H), 7.60-7.56 (m, 1H), 3.57(s, 3H). MS: (ESI, m/z): 376[M+H]⁺.

Example 10-1.3-(5-cyano-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: Synthesis of2-bromo-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrileand2-bromo-3-methyl-6-(trifluoromethyl)-3H-benzo[d]imidazole-5-carbonitrile

Sodium hydride (60% dispersion in mineral oil, 28 mg, 0.70 mmol) wasadded to a 0° C. solution of2-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile (200 mg,0.69 mmol) in DMF (5 mL), and the mixture was stirred for 30 minutes.Methyl iodide (0.7 mL, 11.2 mmol) was added at 0° C., and the resultingsolution stirred for additional 1 h at 0° C. The reaction was quenchedby the addition of 10 mL of water, and the resulting solution wasextracted with 3×10 mL of ethyl acetate. The combined organic phaseswere dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography withsilica gel (eluting with ethyl acetate/petroleum ether (1:2)) to give amixture of2-bromo-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrileand2-bromo-3-methyl-6-(trifluoromethyl)-3H-benzo[d]imidazole-5-carbonitrile(180 mg, 86%) as light yellow oil. MS: (ESI, m/z): 304[M+H]⁺.

Step 2: Synthesis of methyl3-(5-cyano-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoateand methyl3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

A 30-mL microwave tube was charged with the mixture of2-bromo-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrileand2-bromo-3-methyl-6-(trifluoromethyl)-3H-benzo[d]imidazole-5-carbonitrile(65 mg, 0.21 mmol), ethanol (10 mL), 6 M aquoues HCl solution (1 drop),and methyl 3-aminobenzoate (97 mg, 0.64 mmol). The reaction mixture wasirradiated with microwave radiation for 1 h at 150° C. and then cooledto room temperature and concentrated under vacuum. The crude product waspurified by Prep-HPLC with the following conditions: Column: WatersSunfire C18, 19×150 mm; Mobile Phase A: water/0.1% FA; Mobile Phase B:ACN; Flow rate: 28 mL/min; Gradient: 20% B to 50% B in 10 min; 254 nm.The first eluting isomer was collected and concentrated under vacuum togive methyl3-(5-cyano-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(21 mg, 26%) as a white solid. MS: (ESI, m/z): 375[M+H]⁺. The secondeluting isomer was collected and concentrated under vacuum to givemethyl3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(27.6 mg, 34%) as a white solid. MS: (ESI, m/z): 375[M+H]⁺.

Step 3:3-(5-cyano-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.45 mL, 7.3 mmol) and 1 Maqueous sodium hydroxide solution (0.11 mL, 0.11 mmol) were added to a0° C. solution of methyl3-(5-cyano-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(21 mg, 0.06 mmol), in THF/MeOH (4:1, 2 mL), and the resulting solutionstirred for 4 h at room temperature. The pH value of the solution wasadjusted to 6 with 2 M aqueous HCl solution. The crude product waspurified by Prep-HPLC with the following conditions: Column: Water HSSC18, 2.1×50 mm, 1.8 um; Mobile Phase A: water/0.05% TFA; Mobile Phase B:ACN/0.05% TFA; Flow rate: 0.7 mL/min; Gradient: 5% B to 95% B in 2.0min, hold 0.6 min; 254 nm. The collected fraction was lyophilized togive3-(5-cyano-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(2.8 mg, 10%) as an off-white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.23 (s, 1H), 9.58 (s, 1H), 9.07 (s, 1H), 8.18-8.02 (m, 4H), 7.47-7.38(m, 2H), 3.87 (s, 3H). MS: (ESI, m/z): 376[M+H]⁺.

Example 11-1.3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 4-amino-5-bromo-2-(trifluoromethyl)benzonitrile

Bromine (4.73 g, 29.6 mmol) was added dropwise to a 0° C. solution of4-amino-2-(trifluoromethyl)benzonitrile (5.0 g, 26.9 mmol) in methanol(80 mL), and the resulting solution stirred for 3 h at room temperature.The resulting mixture was quenched by the addition of 15 mL of saturatedaqueous NaHSO₃ solution at 0° C. The methanol was removed under vacuum,and the resulting solution was extracted with 3×20 mL ofdichloromethane. The combined organic phases were washed with 3×10 mL ofwater, dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography withsilica gel (eluting with ethyl acetate/petroleum ether (1:3)) to give4-amino-5-bromo-2-(trifluoromethyl)benzonitrile (6.5 g, 91%) as a whitesolid. MS: (ESI, m/z): 263 [M−H]⁻.

Step 2: 4-amino-5-(methylamino)-2-(trifluoromethyl)benzonitrile

A solution of 4-amino-5-bromo-2-(trifluoromethyl)benzonitrile (1.5 g,5.68 mmol), methylamine hydrochloride (3.81 g, 56.82 mmol), cesiumcarbonate (22.23 g, 68.18 mmol), 2,2,6,6-tetramethylheptane-3,5-dione(2.09 g, 11.36 mmol) and copper (I) iodide (1.40 g, 7.34 mmol) in DMSO(45 mL) stirred for 23 h at room temperature. The resulting solution wasdiluted with 30 mL of water and extracted with 3×30 mL ofdichloromethane. The combined organic phases were washed with water (30mL), dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography withsilica gel (eluting with ethyl acetate/petroleum ether (1:4)) to give4-amino-5-(methylamino)-2-(trifluoromethyl)benzonitrile (750 mg, 62%) asa brown solid. MS: (ESI, m/z): 216[M+H]⁺.

Step 3: methyl3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of 4-amino-5-(methylamino)-2-(trifluoromethyl)benzonitrile(200 mg, 0.93 mmol), methyl 3-isothiocyanatobenzoate (270 mg, 1.40mmol), and EDC-HCl (370 mg, 1.86 mmol) in tetrahydrofuran (10 mL)stirred for 5 h at 70° C. The resulting mixture was cooled to roomtemperature and concentrated under vacuum. The residue was dissolved in3 mL of DMF and purified by reversed phase column with the followingconditions: Column: C18 column, 40 g, 20-45 um, 100 A; Mobile Phase:water/0.05% TFA and ACN (2% upto 50% in 30 min); Flow rate: 80 mL/min;254 nm. The collected fraction was concentrated under vacuum to givemethyl3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(60 mg, 17%) as a brown solid. MS: (ESI, m/z): 375[M+H]⁺.

Step 4:3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid

A solution of methyl3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(100 mg, 0.27 mmol) and lithium hydroxide monohydrate (56 mg, 1.33 mmol)in water (5 mL) and THF (5 mL) stirred for 10 h at room temperature. Theresulting mixture was diluted with 10 mL of water and extracted with 10mL of ethyl acetate. The aqueous layer was separated and the pH value ofthe solution was adjusted to 5 with 4 M aqueous HCl solution. Theresulting solution was extracted with 2×20 mL of dichloromethane, andthe combined organic phases were washed with 30 mL of brine, dried overanhydrous sodium sulfate, filtered, and concentrated under vacuum togive3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid (90 mg, 94%) as a yellow solid. MS: (ESI, m/z): 361[M+H]⁺.

Step 5:3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

A solution of3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid (200 mg, 0.56 mmol), IPCF (0.31 g, 2.78 mmol), NMM (250 mg, 2.47mmol), and hydroxylamine hydrochloride (0.11 g, 1.59 mmol) in DMA (2 mL)stirred for 10 h at room temperature. The crude product was purified byPrep-HPLC with the following conditions: Column Waters RP 18, 19×150 mm,5 um; mobile phase, water (0.1% FA)/CH₃CN; Solvent B increase from 20 to41% in 10 min; Detector 220 & 254 nm. The collected fraction waslyophilized to give3-(6-cyano-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(17 mg, 7%) as a light brown solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.22 (br, 1H), 9.71 (br, 1H), 8.24-8.15 (m, 3H), 7.91 (d, J=5.6 Hz,1H), 7.46-7.38 (m, 2H), 3.83 (s, 3H). MS: (ESI, m/z): 376[M+H]⁺.

Example 12-1.3-(6-cyano-5-fluoro-1-methyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 4-amino-2-fluoro-5-nitrobenzonitrile

Ammonium hydroxide (13 mL, 23.9 mmol) was added to a 0° C. solution of2,4-difluoro-5-nitrobenzonitrile (4.41 g, 23.95 mmol) in ethanol (3 mL),and the resulting solution stirred for 3 h at room temperature. Theresulting mixture was filtered and the filter cake was dried to give4-amino-2-fluoro-5-nitrobenzonitrile (4.4 g, 100%) as a yellow solid.MS: (ESI, m/z):182[M+H]⁺.

Step 2: tert-butyl 4-cyano-5-fluoro-2-nitrophenylcarbamate

Sodium hydride (60% dispersion in mineral oil, 1.16 g, 29 mmol) wasadded to a 0° C. solution of 4-amino-2-fluoro-5-nitrobenzonitrile (4.4g, 23.95 mmol) in THF (30 mL), and the solution stirred for 30 min atroom temperature. Di-tert-butyl dicarbonate (6.36 g, 29.1 mmol) wasadded, and the solution stirred overnight at room temperature. Thereaction was quenched by the addition of 20 mL of water, and theresulting solution was extracted with 3×20 mL of ethyl acetate. Thecombined organic phases were washed with 3×20 mL of brine, dried overanhydrous Na₂SO₄, filtered and concentrated under vacuum. The residuewas purified via column chromatography with silica gel (eluting withethyl acetate/petroleum ether (1:4)) to give tert-butyl4-cyano-5-fluoro-2-nitrophenylcarbamate (4.3 g, 63% two steps) as awhite solid. MS: (ESI, m/z):282[M+H]⁺.

Step 3: tert-butyl 2-amino-4-cyano-5-fluorophenylcarbamate

A solution of tert-butyl 4-cyano-5-fluoro-2-nitrophenylcarbamate (4.3 g,15.3 mmol), iron filings (4.28 g, 76.6 mmoL), and ammonium chloride (810mg, 15.1 mmol) in EtOH/H₂O (4:1, 40 mL) stirred for 4 h at 90° C. in anoil bath. The reaction mixture was filtered, and the filtrate wasconcentrated under vacuum. The residue was diluted with 20 mL of waterand extracted with 3×20 mL of ethyl acetate. The combined organic phaseswere washed with 3×20 mL of brine, dried over anhydrous Na₂SO₄, filteredand concentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with dichloromethane/methanol(10:1)) to give tert-butyl 2-amino-4-cyano-5-fluorophenylcarbamate (4 g)as a yellow solid. MS: (ESI, m/z):252[M+H]⁺.

Step 4: tert-butyl 4-cyano-5-fluoro-2-(methylamino)phenylcarbamate

A solution of tert-butyl 2-amino-4-cyano-5-fluorophenylcarbamate (4 g,15.3 mmol), formaldehyde (1.43 g, 47.76 mmol) in MeOH (20 mL) stirredfor 2 h, and then sodium cyanoborohydride (5.00 g, 79.6 mmol) was added.The resulting solution stirred overnight at room temperature, and wasthen quenched by the addition of 20 mL of water. The resulting solutionwas extracted with 3×20 mL of ethyl acetate, and the combined organicphases were washed with 3×20 mL of brine, dried over anhydrous Na₂SO₄,filtered and concentrated under vacuum. The residue was purified viacolumn chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:1)) to give tert-butyl4-cyano-5-fluoro-2-(methylamino)phenylcarbamate (2 g, 49% two steps) asa yellow solid. MS: (ESI, m/z):266[M+H]⁺.

Step 5: 4-amino-2-fluoro-5-(methylamino)benzonitrile

A solution of tert-butyl 4-cyano-5-fluoro-2-(methylamino)phenylcarbamate(2.00 g, 7.54 mmol) in trfluoroacetic acid/dichloromethane (1:10, 30 mL)stirred overnight at room temperature, and the resulting mixture wasconcentrated under vacuum. The residue was dissolved in 10 mL ofdichloromethane, and the pH value of the solution was adjusted to 7 withsaturated aqueous sodium bicarbonate solution. The mixture was extractedwith dichloromethane (3×10 mL), and the combined organic phases weredried over anhydrous Na₂SO₄, filtered and concentrated under vacuum. Theresidue was purified via column chromatography with silica gel (elutingwith dichloromethane/methanol (10:1)) to give4-amino-2-fluoro-5-(methylamino)benzonitrile (1.6 g) as a white solid.MS: (ESI, m/z):166[M+H]⁺.

Step 6: 6-fluoro-2-mercapto-3-methyl-3H-benzo[d]imidazole-5-carbonitrile

A solution of 4-amino-2-fluoro-5-(methylamino)benzonitrile (1.6 g, 7.54mmol), potassium hydroxide (1.629 g, 29.03 mmol), and carbon disulfide(1.47 g, 19.3 mmoL) in EtOH (10 mL) stirred for 4 h at 90° C. in an oilbath. The resulting mixture was cooled to room temperature andconcentrated under vacuum. Water (10 mL) was added and the mixture wasextracted with 3×15 mL of ethyl acetate. The combined organic phaseswere dried over anhydrous Na₂SO₄, filtered and concentrated undervacuum. The residue was purified via column chromatography with silicagel (eluting with ethyl acetate/petroleum ether (1:1)) to give6-fluoro-2-mercapto-3-methyl-3H-benzo[d]imidazole-5-carbonitrile (550mg, 35% two steps) as a brown solid. MS: (ESI, m/z):208[M+H]⁺.

Step 7: 2-bromo-6-fluoro-3-methyl-3H-benzo[d]imidazole-5-carbonitrile

Bromine (0.54 mL, 10.5 mmol) was added dropwise to a 0° C. solution of6-fluoro-2-mercapto-3-methyl-3H-benzo[d]imidazole-5-carbonitrile (547mg, 2.64 mmol) in hydrogen bromide (40% solution in acetic acid, 10 mL),and the resulting solution was stirred for 3 h at 0° C. The reaction wasquenched by the addition of water (15 mL) and cooled to 0° C. The pHvalue of the solution was adjusted to 5 with 2 M aqueous sodiumhydroxide solution, and the resulting solution was extracted with 3×20mL of ethyl acetate. The combined organic phases were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residuewas purified via column chromatography with silica gel (eluting withethyl acetate/petroleum ether (3:2)) to give2-bromo-6-fluoro-3-methyl-3H-benzo[d]imidazole-5-carbonitrile (120 mg,18%) as a yellow solid. MS: (ESI, m/z): 254,256[M+H]⁺.

Step 8: methyl3-(6-cyano-5-fluoro-1-methyl-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of2-bromo-6-fluoro-3-methyl-3H-benzo[d]imidazole-5-carbonitrile (100 mg,0.39 mmol), methyl 3-aminobenzoate (119 mg, 0.79 mmol), and 6 M aqueousHCl solution (1 drop) in EtOH (8 mL). The reaction mixture wasirradiated with microwave radiation for 2 h at 150° C. and then cooledto room temperature. Water (10 mL) was added, and the resulting solutionwas extracted with 3×15 mL of ethyl acetate. The combined organic phaseswere dried over anhydrous Na₂SO₄, filtered, and concentrated undervacuum. The residue was purified via column chromatography with silicagel (eluting with dichloromethane/methanol (20:1)) to give methyl3-(6-cyano-5-fluoro-1-methyl-1H-benzo[d]imidazol-2-ylamino)benzoate (50mg, 39%) as a brown solid. MS: (ESI, m/z):325[M+H]⁺.

Step 9:3-(6-cyano-5-fluoro-1-methyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.45 mL, 7.3 mmol) and 1 Maqueous sodium hydroxide solution (0.24 mL, 0.24 mmol) were added to a0° C. solution of methyl3-(6-cyano-5-fluoro-1-methyl-1H-benzo[d]imidazol-2-ylamino)benzoate (40mg, 0.12 mmol) in THF/MeOH (4:1, 1 mL), and the resulting solutionstirred for 2 h at room temperature and was then cooled to 0° C. The pHvalue of the solution was adjusted to 5 with 6 M aqueous HCl solution.The crude product was purified by Prep-HPLC with the followingconditions: Column: Waters HSS C18, 2.1×50 mm, 1.8 um; Mobile Phase A:Water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 0.7 mL/min; Gradient:5% B to 95% B in 2.0 min, hold 0.6 min; 254 nm. The collected fractionwas lyophilized to give3-(6-cyano-5-fluoro-1-methyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(13 mg, 24%) as a white solid. ¹H-NMR (DMSO, 300 MHz), δ (ppm): 11.23(br, 1H), 9.63 (s, 1H), 8.14-8.07 (m, 2H), 7.92 (d, J=5.7 Hz, 1H),7.47-7.38 (m, 3H), 3.76 (s, 3H). MS: (ESI, m/z): 326[M+H]⁺.

Example 13-1.3-(5,6-dichloro-1-methyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 4,5-dichloro-N-methyl-2-nitrobenzenamine

A solution of 4,5-dichloro-2-nitroaniline (2.00 g, 9.66 mmol), dimethylsulfate (1.4 g, 11.1 mmol), tetrabutylammonium bromide (0.2 g, 0.58mmol), and sodium hydroxide (4.00 g, 100.0 mmol) in toluene (30 mL)stirred for 5 h at room temperature. The reaction mixture was dilutedwith saturated aqueous ammonium chloride solution and extracted with3×50 mL of ethyl acetate. The combined organic phases were dried overanhydrous sodium sulfate, filtered, and concentrated under vacuum. Theresidue was purified via column chromatography with silica gel (elutingwith ethyl acetate/petroleum ether (1:3)) to give4,5-dichloro-N-methyl-2-nitrobenzenamine (1.9 g, 89%) as a white solid.MS: (ESI, m/z): 219[M−H]⁻.

Step 2: 4,5-dichloro-N1-methylbenzene-1,2-diamine

Iron filings (0.76 g, 13.56 mmol) were added in portions to a 60° C.solution of 4,5-dichloro-N-methyl-2-nitrobenzenamine (500 mg, 2.26 mmol)and ammonium chloride (244 mg, 4.56 mmol) in ethanol (30 mL) and water(5 mL). The resulting solution stirred for 2 days at 80° C. in an oilbath. The reaction mixture was cooled to room temperature. The reactionmixture was filtered, and the filtrate was concentrated under vacuum.The residue was diluted with 50 mL of water and extracted with 3×30 mLof ethyl acetate. The combined organic phases were dried over anhydroussodium sulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:2)) to give4,5-dichloro-N1-methylbenzene-1,2-diamine (160 mg, 37%) as a brownsolid. MS: (ESI, m/z): 191[M+H]⁺.

Step 3: methyl3-(5,6-dichloro-1-methyl-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of 4,5-dichloro-N1-methylbenzene-1,2-diamine (100 mg, 0.52mmol methyl 3-isothiocyanatobenzoate (102 mg, 0.53 mmol),N,N′-diisopropylcarbodiimide (132 mg, 1.05 mmol) in THF (50 mL) stirredovernight at 70° C. in an oil bath. The reaction mixture was cooled toroom temperature and then concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:2)) to give methyl3-(5,6-dichloro-1-methyl-1H-benzo[d]imidazol-2-ylamino)benzoate (70 mg,38%) as an off-white solid. MS: (ESI, m/z): 350[M+H]⁺.

Step 4:3-(5,6-dichloro-1-methyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideHydroxyl amine solution (50% in water, 0.53 mL, 8.6 mmol) and 1 Maqueous sodium hydroxide solution (0.57 mL, 0.57 mmol) were added to asolution of methyl3-(5,6-dichloro-1-methyl-1H-benzo[d]imidazol-2-ylamino)benzoate (100 mg,0.29 mmol) in THF/MeOH (4:1, 2 mL), and the resulting solution stirredfor 1 h at room temperature. The pH value of the resulting solution wasadjusted to 6 with 6 M aqueous HCl solution. The solids were collectedby filtration and recrystallized from 2 mL of MeOH to give3-(5,6-dichloro-1-methyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(58 mg, 58%) as a off-white solid. ¹H-NMR (DMSO, 300 MHz), δ (ppm):11.18 (s, 1H), 9.29 (s, 1H), 9.02 (s, 1H), 8.03 (d, J=5.7 Hz, 2H), 7.63(d, J=20.7 Hz, 2H), 7.43-7.31 (m, 2H), 3.72 (s, 3H). MS: (ESI, m/z): 351[M+H]⁺.

Example 14-1.3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 4-amino-5-(isopropylamino)-2-(trifluoromethyl)benzonitrile

A solution of 4-amino-5-bromo-2-(trifluoromethyl)benzonitrile (100 mg,0.38 mmol), propan-2-amine (230 mg, 3.89 mmol), copper (I) iodide (80mg, 0.42 mmol), cesium carbonate (370 mg, 1.14 mmol) and2,2,6,6-tetramethylheptane-3,5-dione (140 mg, 0.76 mmol) in DMSO (2 mL)stirred for 15 h at room temperature. The resulting mixture wasconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:10)) to give4-amino-5-(isopropylamino)-2-(trifluoromethyl)benzonitrile (30 mg, 33%)as a pink solid. MS: (ESI, m/z): 244[M+H]⁺.

Step 2: methyl3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of 4-amino-5-(isopropylamino)-2-(trifluoromethyl)benzonitrile(300 mg, 1.23 mmol), methyl 3-isothiocyanatobenzoate (470 mg, 2.45mmol), and EDC-HCl (708 mg, 3.69 mmol) in THF (30 mL) stirred for 10 hat 70° C. The resulting mixture was cooled to room temperature andconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:1)) to give methyl3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(200 mg, 40%) as a brown solid. MS: (ESI, m/z): 403[M+H]⁺.

Step 3:3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid

A solution of methyl3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(100 mg, 0.25 mmol), lithium hydroxide monohydrate (50 mg, 1.24 mmol) inwater (4 mL) and THF (5 mL) stirred for 10 h at room temperature. Theresulting mixture was diluted with 10 mL of water and extracted with 10mL of ethyl acetate. The aqueous layer was separated and the pH value ofthe solution was adjusted to 5 with 4 M aqueous HCl solution. Theresulting solution was extracted with 2×20 mL of dichloromethane, andthe combined organic phases were washed with 30 mL of brine, dried overanhydrous sodium sulfate, filtered, and concentrated under vacuum togive3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid (80 mg, 83%) as a yellow solid. MS: (ESI, m/z): 389[M+H]⁺.

Step 4:3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

A solution of3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid (80 mg, 0.21 mmol), IPCF (129 mg, 1.03 mmol), NMM (104 mg, 1.03mmol) and hydroxyl amine hydrochloride (71 mg, 1.03 mmol) in DMA (3 mL)stirred for 10 h at room temperature. The crude product was purified byprep-HPLC with the following conditions: Column: XBridge RP C18, 19×150mm, 5 um; Mobile Phase A: water/0.05% TFA; Mobile Phase B: ACN; Flowrate: 25 mL/min; Gradient: 20% B to 39% B in 10 min, 254 nm. Thecollected fraction was lyophilized to give3-(6-cyano-1-isopropyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(37.2 mg, 45%) as an off-white solid. ¹H-NMR (DMSO, 300 MHz), δ (ppm):11.20 (br, 1H), 9.51 (br, 1H), 8.33 (s, 1H), 8.08-8.03 (m, 2H), 7.86 (s,1H), 7.42-7.34 (m, 2H), 5.04-4.95 (m, 1H), 1.58 (d, J=6.9 Hz, 6H). MS:(ESI, m/z): 404[M+H]⁺.

Example 15-1.3-(5-cyano-1-isopropyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 5-bromo-4-(isopropylamino)-2-(trifluoromethyl)benzonitrile

Trifluoroacetic acid (0.60 mL, 7.96 mmol) was added dropwise to asolution of 4-amino-5-bromo-2-(trifluoromethyl)benzonitrile (2.0 g, 7.55mmol) and 2,2-dimethoxypropane (3.92 g, 37.64 mmol) in toluene (20 mL),and the mixture stirred for 1 h at room temperature. The reactionmixture was cooled to 0° C., and borane dimethyl sulfide complex (10 M,0.83 mL, 8.31 mmol) was added dropwise. The resulting solution stirredfor 18 h at room temperature, and was then added dropwise into 40 mL ofmethanol. The resulting mixture was concentrated under vacuum. Theresidue was purified via column chromatography with silica gel (elutingwith ethyl acetate/petroleum ether (1:7)) to give5-bromo-4-(isopropylamino)-2-(trifluoromethyl)benzonitrile (1.583 g,68%) as a light brown solid. MS: (ESI, m/z): 305[M−H]⁻.

Step 2: 5-amino-4-(isopropylamino)-2-(trifluoromethyl)benzonitrile

A solution of 5-bromo-4-(isopropylamino)-2-(trifluoromethyl)benzonitrile(1.0 g, 3.26 mmol), pentane-2,4-dione (651 mg, 6.51 mmol), copper (II)acetylacetonate (0.426 g, 1.63 mmol), cesium carbonate (2.12 g, 6.51mmol), ammonium hydroxide (0.30 mL, 16 mmol) in DMF (15 mL) stirred for1 day at 90° C. The resulting solution was cooled to room temperature,diluted with 20 mL of water, and extracted with 3×30 mL of ethylacetate. The combined organic phases were dried over anhydrous sodiumsulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:3)) to give5-amino-4-(isopropylamino)-2-(trifluoromethyl)benzonitrile (669 mg, 84%)as a blue green oil. MS: (ESI, m/z): 244[M+H]⁺.

Step 3: methyl3-(5-cyano-1-isopropyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

EDC-HCl (790 mg, 4.12 mmol) was added to a 0° C. solution of5-amino-4-(isopropylamino)-2-(trifluoromethyl)benzonitrile (400 mg, 1.64mmol) and methyl 3-isothiocyanatobenzoate (477 mg, 2.47 mmol) in THF (35mL), and the resulting solution was stirred for 16 h at 75° C. Themixture was cooled to room temperature and concentrated under vacuum.The residue was diluted with 40 mL of ethyl acetate and washed with 3×20mL of water. The combined organic phases were dried over anhydroussodium sulfate and concentrated under vacuum. The crude product waspurified by reversed phase column with the following conditions: Column:C18 column, 40 g, 20-45 um, 100 A; Mobile Phase: water and ACN (5% up to50% in 30 min); Flow rate: 80 mL/min; 254 nm. The collected fraction wasconcentrated under vacuum to give methyl3-(5-cyano-1-isopropyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(265.9 mg, 40%) as a yellow solid. MS: (ESI, m/z): 403[M+H]⁺.

Step 4:3-(5-cyano-1-isopropyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.59 mL, 9.7 mmol) and 1 Maqueous sodium hydroxide solution (0.65 mL, 0.65 mmol) were added to asolution of methyl3-(5-cyano-1-isopropyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(130 mg, 0.32 mmol) in THF/MeOH (4:1, 2 mL), and the resulting solutionstirred for 3 h at room temperature. The crude product was purified byPrep-HPLC with the following conditions: Column: Xbridge RP18 19×150, 5um, 19×100 mm; Mobile phase: water with 0.05% TFA and ACN (4% ACN up to58% in 7 min); Flow rate: 25 ml/min; Detector: 254, 220 nm. Thecollected fraction was lyophilized to give3-(5-cyano-1-isopropyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(10.7 mg, 8%) as an off-white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.20 (s, 1H), 9.46 (s, 1H), 8.11-8.05 (m, 3H), 7.96 (s, 1H), 7.46-7.38(m, 2H), 5.10-5.03 (m, 1H), 1.62 (d, J=6.8 Hz, 6H). MS: (ESI, m/z):404[M+H]⁺.

Example 16-1.N-hydroxy-3-(1-isopropyl-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide

Step 1: N-(3-bromo-4-(trifluoromethyl)phenyl)acetamide

A solution of 3-bromo-4-(trifluoromethyl)aniline (10 g, 41.7 mmol) inacetic anhydride (50 mL) stirred for 2 h at room temperature, and theresulting mixture was concentrated under vacuum. The crude product wasre-crystallized from 20 mL of ethyl acetate-petroleum ether (1/10) togive N-(3-bromo-4-(trifluoromethyl)phenyl)acetamide (9.4 g, 80%) as awhite solid. MS: (ESI, m/z): 280[M−H]0.325[M−H]⁻.

Step 2: N-(5-bromo-2-nitro-4-(trifluoromethyl)phenyl)acetamide

Concentrated nitric acid (4.2 mL) was added dropwise to a 0° C. solutionof N-(3-bromo-4-(trifluoromethyl)phenyl)acetamide (8.4 g, 29.8 mmol) inconcentrated sulfuric acid (33.6 mL), and the resulting solution stirredfor 10 min at room temperature. The reaction was then added dropwiseinto 1000 mL of ice/water. The solids were collected by filtration andwashed with 500 mL of water and dried under vacuum to giveN-(5-bromo-2-nitro-4-(trifluoromethyl)phenyl)acetamide (8.4 g, 86%) as ayellow solid. MS: (ESI, m/z): 325[M−H]⁻.

Step 3: N-(4-nitro-6-(trifluoromethyl)biphenyl-3-yl)

A solution of N-(5-bromo-2-nitro-4-(trifluoromethyl)phenyl)acetamide(8.6 g, 26.3 mmol), phenylboronic acid (6.4 g, 52 mmol), Pd(dppf)Cl₂dichloromethane adduct (1.07 g, 1.31 mmol), and potassium carbonate(10.9 g, 78.8 mmol) in water (20 mL) and 1,4-dioxane (100 mL) stirredovernight at 90° C. The resulting mixture was cooled to room temperatureand concentrated under vacuum. The residue was diluted with 200 mL ofwater and extracted with 2×100 mL of ethyl acetate. The combined organicphases were washed with 1×200 mL of brine, dried over anhydrous sodiumsulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:5)) to giveN-(4-nitro-6-(trifluoromethyl)biphenyl-3-yl) (5.4 g, 63%) as a yellowsolid. MS: (ESI, m/z): 323 [M−H]⁻.

Step 4: 4-nitro-6-(trifluoromethyl)biphenyl-3-amine

A solution of N-(4-nitro-6-(trifluoromethyl)biphenyl-3-yl)acetamide (5.4g, 16.65 mmol) in 1,4-dioxane (50 mL) and 6 M aqueous HCl solution (25mL) stirred for 3 h at 90° C. The resulting mixture was cooled to roomtemperature and concentrated under vacuum. The residue was diluted with100 mL of 1 M aqueous NaHCO₃ solution, and the resulting solution wasextracted with 2×150 mL of ethyl acetate. The combined organic phaseswere washed with 1×200 mL of brine, dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum. The residue was purified viacolumn chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:5)) to give4-nitro-6-(trifluoromethyl)biphenyl-3-amine (4.6 g, 98%) as a yellowsolid. MS: (ESI, m/z): 281[M−H]⁻.

Step 5: N-isopropyl-4-nitro-6-(trifluoromethyl)biphenyl-3-amine

Sodium hydride (60% dispersion in mineral oil, 85 mg, 2.13 mmol) wasadded in portions to a 0° C. solution of4-nitro-6-(trifluoromethyl)biphenyl-3-amine (200 mg, 0.71 mmol) in DMF(5 mL), and the resulting solution stirred for 1 h at room temperature.2-Iodopropane (241 mg, 1.42 mmol) was added dropwise, and the resultingsolution stirred overnight at room temperature. The reaction mixture waspoured into 50 mL of ice/water and extracted with 2×50 mL of ethylacetate. The combined organic phases were washed with 2×100 mL of brine,dried over anhydrous sodium sulfate, filtered, and concentrated undervacuum. The residue was purified via column chromatography with silicagel (eluting with ethyl acetate/petroleum ether (1:5)) to giveN-isopropyl-4-nitro-6-(trifluoromethyl)biphenyl-3-amine (140 mg, 61%) asa yellow solid. MS: (ESI, m/z): 323[M−H]⁻.

Step 6: N3-isopropyl-6-(trifluoromethyl)biphenyl-3,4-diamine

A mixture of N-isopropyl-4-nitro-6-(trifluoromethyl)biphenyl-3-amine(800 mg, 2.47 mmol) and 10% platinum on carbon (150 mg) in methanol (50mL) stirred for 2 h at room temperature under a hydrogen atmosphere. Thereaction mixture was filtered, and the filtrate was concentrated undervacuum to give N3-isopropyl-6-(trifluoromethyl)biphenyl-3,4-diamine (670mg, 92%) as yellow oil. MS: (ESI, m/z): 295[M+H]⁺.

Step 7: methyl3-(1-isopropyl-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of N3-isopropyl-6-(trifluoromethyl)biphenyl-3,4-diamine (200mg, 0.68 mmol) and methyl 3-isothiocyanatobenzoate (144 mg, 0.75 mmol)in THF (20 mL) stirred for 2 h at room temperature. CDI (214 mg, 1.32mmol) was added, and the resulting solution stirred overnight at 75° C.in an oil bath. The resulting mixture was cooled to room temperature andconcentrated under vacuum. The crude product was purified by preparativethin layer chromatography (eluting with ethyl acetate/petroleum ether(1:2)). The collected fraction was concentrated under vacuum to givemethyl3-(1-isopropyl-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(90 mg, 30%) as a yellow oil. MS: (ESI, m/z): 454[M+H]⁺.

Step 8:N-hydroxy-3-(1-isopropyl-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide

Hydroxyl amine solution (50% in water, 0.73 mL, 11.9 mmol) and 1 Maqueous sodium hydroxide solution (0.40 mL, 0.40 mmol) were added to asolution of methyl3-(1-isopropyl-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(90 mg, 0.20 mmol) in THF/MeOH (3:1, 3 mL), and the resulting solutionstirred for 2 h at room temperature. The solids were filtered out. Thecrude product was purified by prep-HPLC with the following conditions:Sunfire C18 Column 19*150 mn; mobile phase, water/0.05% TFA and CH₃CN(5% B upto 55% B in 1.1 min;); Flow rate: 25 mL/min; Detector, 254 nm.The collected fraction was lyophilized to giveN-hydroxy-3-(1-isopropyl-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide(36.0 mg, 32%) as a pink solid. ¹H-NMR (CD₃OD, 400 MHz), δ (ppm): 7.98(d, J=1.4 Hz, 1H), 7.79 (s, 1H), 7.73-7.71 (m, 2H), 7.67-7.64 (m, 2H),7.47-7.44 (m, 3H), 7.39-7.38 (m, 2H), 4.98-4.91 (m, 1H), 1.75 (d, J=6.8Hz, 6H). MS: (ESI, m/z): 455[M+H]⁺.

The following compounds were prepared according to the proceduresoutlined above forN-hydroxy-3-(1-isopropyl-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide.

(ESI, m/z) Ex. Structure Name ¹H NMR [M + H]⁺ 16-2

N-hydroxy-3-((1- isopropyl-5-phenyl- 6-(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl) amino)benzamide (DMSO, 400 MHz, ppm): 11.19 (br, 1H),9.48 (br, 1H), 8.08-8.02 (m, 2H), 7.84 (s, 1H), 7.45- 7.32 (m, 8H),5.07- 5.00 (m, 1H), 1.64 (d, J = 6.8 Hz, 6H). 455

Example 17-1.3-(5,6-dichloro-1-isopropyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 1,2,4-trichloro-5-nitrobenzene

1,2,4-Trichlorobenzene (5.00 g, 27.6 mmol) was added dropwise to a 0° C.solution of nitric acid (98%, 18 mL) and water (2 mL), and the resultingsolution stirred for 3 h at room temperature. The reaction mixture wasthen added dropwise into 30 mL of ice/water and the resulting mixturewas filtered. The filter cake was washed with water and dried undervacuum to give 1,2,4-trichloro-5-nitrobenzene (4.9 g, 79%) as a yellowgreen solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm): 8.52 (d, J=4.4 Hz, 1H),8.28 (d, J=4.4 Hz, 1H).

Step 2: 4,5-dichloro-N-isopropyl-2-nitrobenzenamine

A solution of 1,2,4-trichloro-5-nitrobenzene (4.9 g, 21.6 mmol),triethylamine (4.5 mL, 32.6 mmol) and propan-2-amine (6.75 g, 114.19mmol) in THF (20 mL) stirred for 15 h at 65° C. in an oil bath. Theresulting mixture was cooled to room temperature and concentrated undervacuum. The residue was purified via column chromatography with silicagel (eluting with ethyl acetate/petroleum ether (1:10)) to give4,5-dichloro-N-isopropyl-2-nitrobenzenamine (3.91 g, 73%) as an orangesolid. ¹H-NMR (CDCl₃, 400 MHz) δ (ppm): 8.27 (s, 1H), 7.92 (d, J=4.4 Hz,1H), 6.97 (s, 1H), 3.81-3.73 (m, 1H), 1.34 (d, J=6.4 Hz, 6H).

Step 3: 4,5-dichloro-N1-isopropylbenzene-1,2-diamine

Zinc (5.12 g, 78.8 mmol) was added in portions to a 0° C. solution of4,5-dichloro-N-isopropyl-2-nitrobenzenamine (3.91 g, 15.7 mmol) inethanol (15 mL) and ammonium hydroxide (20 mL), and the resultingsolution stirred for 5 h at room temperature. The solids were filteredout, and the resulting solution was extracted with 3×100 mL of ether.The combined organic phases were washed with 100 mL of brine, dried overanhydrous sodium sulfate, filtered, and concentrated under vacuum. Theresidue was purified via column chromatography with silica gel (elutingwith ethyl acetate/petroleum ether (1:10)) to give4,5-dichloro-N1-isopropylbenzene-1,2-diamine (1.03 g, 30%) as a blackoil. MS: (ESI, m/z): 219[M+H]⁺.

Step 4: methyl3-(5,6-dichloro-1-isopropyl-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of 4,5-dichloro-N1-isopropylbenzene-1,2-diamine (300 mg, 1.37mmol) and methyl 3-isothiocyanatobenzoate (292 mg, 1.51 mmol) in THF (20mL) stirred for 3 h at room temperature. N,N′-Diisopropylcarbodiimide(347 mg, 2.75 mmol) was added, and the resulting solution stirred for 16h at 80° C. in an oil bath. The reaction mixture was cooled to roomtemperature and concentrated under vacuum. The residue was purified byreversed phase column with the following conditions: Column: C18 column,40 g, 20-45 um, 100 A; Mobile phase: water with 0.05% NH₄HCO₃ and CAN(40% up to 50% in 30 minutes), 254 & 220 nm. The collected fraction wasconcentrated under vacuum to give methyl3-(5,6-dichloro-1-isopropyl-1H-benzo[d]imidazol-2-ylamino)benzoate (190mg, 37%) as a pink solid. MS: (ESI, m/z): 378[M+H]⁺.

Step 5:3-(5,6-dichloro-1-isopropyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideHydroxyl amine solution (50% in water, 0.97 mL, 15.9 mmol) and 1 Maqueous sodium hydroxide solution (0.53 mL, 0.53 mmol) were added to asolution of methyl3-(5,6-dichloro-1-isopropyl-1H-benzo[d]imidazol-2-ylamino)benzoate (100mg, 0.26 mmol) in THF/MeOH (4:1, 2 mL), and the resulting solutionstirred for 4 h at room temperature. The crude product was purified byprep-HPLC with the following conditions: Column, XBridge RP C18, 19×150mm, 5 um; Mobile Phase A: Water/0.05% TFA; Mobile Phase B: ACN; Flowrate: 25 mL/min; Gradient: 5% B to 50% B in 7.0 min, 254 nm. Thecollected fraction was lyophilized to give3-(5,6-dichloro-1-isopropyl-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(59.3 mg, 45%) as an off-white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.20 (br, 1H), 9.65 (br, 1H), 8.04-7.85 (m, 3H), 7.60 (s, 1H),7.48-7.39 (m, 2H), 4.95-4.91 (m, 1H), 1.57 (d, J=6.8 Hz, 6H). MS: (ESI,m/z): 379[M+H]⁺.

Example 18-1.3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 4-amino-5-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile

A solution of 4-amino-5-bromo-2-(trifluoromethyl)benzonitrile (500 mg,1.89 mmol), 2-methoxyethylamine (1.40 g, 18.6 mmol), cesium carbonate(1.25 g, 3.84 mmol), copper (I) iodide (360 mg, 1.89 mmol), and2,2,6,6-tetramethylheptane-3,5-dione (500 mg, 2.71 mmol) in DMSO (16 mL)stirred for 18 h at room temperature. The reaction mixture was thenpoured into water (100 mL) and extracted with 3×30 mL of ethyl acetate.The organic phase was separated and washed with 10×30 mL of water andconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:1)) give4-amino-5-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile (200 mg,41%) as a brown solid. MS: (ESI, m/z): 258[M−H]⁻.

Step 2: methyl3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution of4-amino-5-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile (300 mg,1.16 mmol), methyl 3-isothiocyanatobenzoate (267 mg, 1.38 mmol), and DIC(360 mg, 2.85 mmol) in THF (10 mL) stirred for 5.5 h at 70° C. Theresulting mixture was cooled to room temperature and concentrated undervacuum. The residue was purified via column chromatography with silicagel (eluting with ethyl acetate/petroleum ether (1:1)) to give methyl3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(70 mg, 14%) as a brown oil. MS: (ESI, m/z): 419[M+H]⁺.

Step 3:3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid

A solution of lithium hydroxide (57.7 mg, 2.41 mmol) in water (0.5 mL)was added dropwise to a 0° C. solution of methyl3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(48 mg, 0.11 mmol) in THF (3 mL), and the reaction mixture stirred for26 h at room temperature. The resulting solution was concentrated undervacuum. The residue was purified by reversed phase column with thefollowing conditions: Column: C18 column, 40 g, 20-45 um, 100 A; Mobilephase: water and ACN (5% up to 40% in 30 minutes), 254 & 220 nm. Thecollected fraction was lyophilized to give3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid (26 mg, 56%) as a white solid. MS: (ESI, m/z): 405[M+H]⁺.

Step 4:3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

A solution of3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoicacid (100 mg, 0.25 mmol), IPCF (160 mg, 1.27 mmol), NMM (130 mg, 1.27mmol) and hydroxylamine hydrochloride (85 mg, 1.27 mmol) in DMA (3 mL)stirred for 10 h at room temperature. The crude product was purified byPrep-HPLC with the following conditions: Column: Waters HSS C18, 2.1×50mm, 1.8 um; Mobile Phase A: water/0.05% TFA, Mobile Phase B: ACN; Flowrate: 0.7 mL/min; Gradient:5% B to 95% B in 2.0 min, hold 0.6 min; 254nm; Detector). The collected fraction was lyophilized to give3-(6-cyano-1-(2-methoxyethyl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(20.8 mg, 20%) as an off-white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.22 (br, 1H), 9.53 (s, 1H), 8.18-8.14 (m, 3H), 7.90 (s, 1H), 7.46-7.38(m, 2H), 4.59-4.57 (m, 2H), 3.70-3.68 (m, 2H), 3.23 (s, 3H). MS: (ESI,m/z): 420[M+H]⁺.

Example 19-1.3-(5-cyano-1-(2-methoxyethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Step 1: 4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile

A solution of 4-amino-2-(trifluoromethyl)benzonitrile (2.00 g, 10.7mmol), potassium iodide (1.79 g, 10.8 mmol), cesium carbonate (10.51 g,32.26 mmol) and 1-bromo-2-methoxyethane (7.47 g, 53.7 mmol) inacetonitrile (25 mL) stirred for 18 h at 70° C. The resulting mixturewas cooled to room temperature and concentrated under vacuum. Theresidue was diluted with water (30 mL) and extracted with 3×30 mL ofethyl acetate. The combined organic phases were washed with 2×30 mL ofbrine, dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography withsilica gel (eluting with ethyl acetate/petroleum ether (1:3)) to give4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile (2.1 g, 80%) asyellow oil. MS: (ESI, m/z): 243 [M−H]⁻.

Step 2: 5-bromo-4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile

Bromine (0.58 mL, 8.6 mmol) was added dropwise to a 0° C. solution of4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile (2.1 g, 8.6mmol) in methanol (30 mL), and the reaction mixture stirred for 4 h atroom temperature. The resulting solution was quenched by the addition of30 mL of saturated aqueous NaHSO₃ solution and the methanol was removedunder vacuum. The resulting solution was extracted with 3×60 mL ofdichloromethane, and the combined organic phases were washed with 2×50mL of water, dried over anhydrous magnesium sulfate, filtered, andconcentrated under vacuum. The residue was purified by reversed phasecolumn with the following conditions: Column: C18 column, 40 g, 20-45um, 100 A; Mobile phase: water and ACN (5% up to 50% in 30 minutes), 254& 220 nm. The collected fraction was concentrated under vacuum to give5-bromo-4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile (1.539g, 55%) as a light yellow solid. MS: (ESI, m/z): 321[M−H]⁻.

Step 3: 5-amino-4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile

A solution of5-bromo-4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile (1.0 g,3.1 mmol), pentane-2,4-dione (619 mg, 6.18 mmol), copper (II)acetylacetonate (405 mg, 1.55 mmol), cesium carbonate (2.017 g, 6.19mmol), ammonium hydroxide (1.2 mL, 15.44 mmol) in DMF (25 mL) stirredfor 2 days at 90° C. The resulting solution was diluted with 25 mL ofwater and extracted with 3×30 mL of ethyl acetate. The combined organicphases were dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified via column chromatography with silicagel (eluting with ethyl acetate/petroleum ether (1:1)) to give5-amino-4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile (255 mg,32%) as a black oil. MS: (ESI, m/z): 258[M−H]⁻.

Step 4: methyl3-(5-cyano-1-(2-methoxyethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

EDC-HCl (47 mg, 0.25 mmol) was added to a 0° C. solution of5-amino-4-(2-methoxyethylamino)-2-(trifluoromethyl)benzonitrile (255 mg,0.98 mmol) and methyl 3-isothiocyanatobenzoate (285 mg, 1.47 mmol) inTHF (30 mL), and the reaction stirred for 6 h at 75° C. The resultingmixture was cooled to room temperature and concentrated under vacuum.The residue was dissolved in 25 mL of ethyl acetate and washed with 2×15mL of water. The combined organic phases were dried over anhydroussodium sulfate, filtered, and concentrated under vacuum. The residue waspurified by reversed phase column with the following conditions: Column:C18 column, 40 g, 20-45 um, 100 A; Mobile phase: water and ACN (5% up to60% in 30 minutes), 254 & 220 nm. The collected fraction wasconcentrated under vacuum to give methyl3-(5-cyano-1-(2-methoxyethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(195 mg, 47%) as a yellow oil. MS: (ESI, m/z): 419 [M+H]⁺.

Step 5:3-(5-cyano-1-(2-methoxyethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.86 mL, 14 mmol) and 1 M aqueoussodium hydroxide solution (0.93 mL, 0.93 mmol) were added to a solutionof methyl3-(5-cyano-1-(2-methoxyethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(195 mg, 0.47 mmol) in THF/MeOH (4:1, 2 mL), and the resulting solutionstirred for 3 h at room temperature. The crude product was purified byprep-HPLC with the following conditions: Column: Xbridge RP18 19×150;mobile phase: water with 0.05% TFA and ACN (5% ACN up to 59% in 7 min);Detector: 254, 220 nm. The collected fraction was lyophilized to give3-(5-cyano-1-(2-methoxyethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamide(12 mg, 6%) as an off-white solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm):11.22 (s, 1H), 9.46 (s, 1H), 9.05 (s, 1H), 8.18-8.09 (m, 3H), 7.99 (s,1H), 7.47-7.38 (m, 2H), 4.62-4.60 (m, 2H), 3.69-3.67 (m, 2H), 3.22 (s,3H). MS: (ESI, m/z): 420[M+H]⁺.

Example 20-1.N-hydroxy-3-(1-(2-methoxyethyl)-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide

Step 1: N-(2-methoxyethyl)-4-nitro-6-(trifluoromethyl)biphenyl-3-amine

Sodium hydride (60% dispersion in mineral oil, 709 mg, 17.9 mmol) wasadded in portions to a 0° C. solution of4-nitro-6-(trifluoromethyl)biphenyl-3-amine (1.00 g, 3.54 mmol) in DMF(50 mL). The reaction mixture stirred for 1 h at room temperature andwas then cooled to 0° C. 2-Bromoethyl methyl ether (1.66 g, 17.9 mmol)was added dropwise, and the resulting solution was stirred overnight atroom temperature. The reaction was poured into 100 mL of ice/water andextracted with 3×100 mL of ethyl acetate. The combined organic phaseswere washed with 3×100 mL of brine, dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum. The residue was purified viacolumn chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:10)) to giveN-(2-methoxyethyl)-4-nitro-6-(trifluoromethyl)biphenyl-3-amine (1 g,83%) as a yellow solid. MS: (ESI, m/z): 341[M+H]⁺.

Step 2: N3-(2-methoxyethyl)-6-(trifluoromethyl)biphenyl-3,4-diamine

A mixture ofN-(2-methoxyethyl)-4-nitro-6-(trifluoromethyl)biphenyl-3-amine (1 g,2.94 mmol) and 10% palladium on carbon (150 mg) in methanol (50 mL)stirred under an atmosphere of hydrogen for 2 h at room temperature. Thereaction mixture was filtered, and the filtrate was concentrated undervacuum to giveN3-(2-methoxyethyl)-6-(trifluoromethyl)biphenyl-3,4-diamine (900 mg,crude) as yellow oil. MS: (ESI, m/z): 311[M+H]⁺.

Step 3: methyl3-(1-(2-methoxyethyl)-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

A solution ofN3-(2-methoxyethyl)-6-(trifluoromethyl)biphenyl-3,4-diamine (200 mg,0.64 mmol), methyl 3-isothiocyanatobenzoate (137 mg, 0.71 mmol), and CDI(203 mg, 1.25 mmol) in THF (20 mL) stirred overnight at 75° C. Theresulting solution was cooled to room temperature, poured into 50 mL ofwater, and extracted with 3×50 mL of ethyl acetate. The combined organicphases were washed with 100 mL of brine, dried over anhydrous sodiumsulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:1)) to giveN3-(2-methoxyethyl)-6-(trifluoromethyl)biphenyl-3,4-diamine (200 mg,66%) as yellow oil. MS: (ESI, m/z): 470[M+H]⁺.

Step 4:N-hydroxy-3-(1-(2-methoxyethyl)-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide

Hydroxyl amine solution (50% in water, 0.78 mL, 12.8 mmol) and 1 Maqueous sodium hydroxide solution (0.42 mL, 0.42 mmol) were added to asolution of methyl3-(1-(2-methoxyethyl)-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(100 mg, 0.21 mmol) in THF/MeOH (4:1, 3 mL), and the resulting solutionstirred for 2 h at room temperature. The solids were filtered out. Thecrude product was purified by prep-HPLC with the following conditions:Sunfire C18 Column 19*150mn; mobile phase, water with 0.05% TFA andCH₃CN (25 mL/min, 5-55% B within 1.1 min); Detector, 254 nm. Thecollected fraction was lyophilized to giveN-hydroxy-3-(1-(2-methoxyethyl)-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide(72.4 mg, 72%) as a pink solid. ¹H-NMR (DMSO, 400 MHz), δ (ppm): 11.23(s, 1H), 9.42 (s, 1H), 8.17-8.12 (m, 2H), 7.76 (s, 1H), 7.48-7.35 (m,8H), 4.53-4.51 (m, 2H), 3.68-3.65 (m, 2H), 3.23 (s, 3H). MS: (ESI, m/z):471[M+H]⁺.

The following compound were prepared according to the proceduresoutlined above forN-hydroxy-3-(1-(2-methoxyethyl)-6-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzamide

(ESI, m/z) Ex. Structure Name ¹HNMR [M + H]⁺ 20-2

N-hydroxy-3-((1- (2-methoxyethyl)- 5-phenyl-6- (trifluoromethyl)-1H-benzo[d] imidazol-2-yl) amino)benzamide (DMSO, 400 MHz, ppm): 11.22(br, 1H), 9.56 (br, 1H), 8.09 (s, 2H), 7.87 (s, 1H), 7.48-7.38 (m, 5H),7.34-7.25 (m, 3H), 4.59-4.70 (m, 2H), 3.73-3.71 (m, 2H), 3.28 (s, 3H)471

Example 21-1.3-(1-(5-aminopentyl)-5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideformate

Step 1: tert-butyl5-(4-cyano-3-(trifluoromethyl)phenylamino)pentylcarbamate

A solution of 4-fluoro-2-(trifluoromethyl)benzonitrile (7.00 g, 37.1mmol), tert-butyl N-(5-aminopentyl)carbamate (10 g, 49.43 mmol), andN,N-diisopropylethyl amine (11 g, 85.11 mmol) in DMF (100 mL) stirredfor 6 h at room temperature. The resulting solution was diluted with 400mL of ethyl acetate, washed with 2×200 mL brine, dried over anhydroussodium sulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:1)) to give tert-butyl5-(4-cyano-3-(trifluoromethyl)phenylamino)pentylcarbamate (11 g, 60%) ascolorless oil. MS: (ESI, m/z): 372[M+H]⁺.

Step 2: tert-butyl5-(2-bromo-4-cyano-5-(trifluoromethyl)phenylamino)pentylcarbamate

A solution of tert-butyl5-(4-cyano-3-(trifluoromethyl)phenylamino)pentylcarbamate (6.000 g,16.16 mmol) and NBS (3.450 g, 19.38 mmol) in DMF (30 mL) stirred for 2 hat room temperature. The resulting solution was poured into 100 mL ofwater and extracted with 2×100 mL of ethyl acetate. The combined organicphases were washed with 3×200 mL of brine, dried over anhydrous sodiumsulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:2)) to give tert-butyl5-(2-bromo-4-cyano-5-(trifluoromethyl)phenylamino)pentylcarbamate (6 g,82%) as a colorless solid. MS: (ESI, m/z): 450[M+H]⁺.

Step 3: tert-butyl5-(2-amino-4-cyano-5-(trifluoromethyl)phenylamino)pentylcarbamate

A solution of tert-butyl5-(2-bromo-4-cyano-5-(trifluoromethyl)phenylamino)pentylcarbamate (3.00g, 6.67 mmol), copper(II) acetylacetonate (1.74 g, 6.67 mmol), andammonium hydroxide (15 mL) in DMF (15 mL) stirred for 16 h at 90° C. inan oil bath. The resulting solution was cooled to room temperature andthen diluted with 200 mL of ethyl acetate. The resulting solution waswashed with 3×100 mL of brine, dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum. The residue was purified viacolumn chromatography with silica gel (eluting with ethylacetate/petroleum ether (1:1)) to give tert-butyl5-(2-amino-4-cyano-5-(trifluoromethyl)phenylamino)pentylcarbamate (2.4g, 93%) as a purple solid. MS: (ESI, m/z): 387[M+H]⁺.

Step 4: methyl3-(1-(5-(tert-butoxycarbonylamino)pentyl)-5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate

Methyl 3-isothiocyanatobenzoate (550 mg, 2.85 mmol) was added to asolution of tert-butyl5-(2-amino-4-cyano-5-(trifluoromethyl)phenylamino)pentylcarbamate (1.00g, 2.59 mmol) in DMF (5 mL), and the reaction mixture stirred for 16 hat room temperature. DIC (490 mg, 3.89 mmol) was added, and theresulting solution stirred for 3 h at 80° C. The reaction mixture wascooled to room temperature, diluted with 100 mL of ethyl acetate, washedwith 2×50 mL of brine, dried over anhydrous sodium sulfate, filtered,and concentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:1)) to give methyl3-(1-(5-(tert-butoxycarbonylamino)pentyl)-5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(850 mg, 60%) as a brown solid. MS: (ESI, m/z): 546[M+H]⁺.

Step 5:3-(1-(5-aminopentyl)-5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideformate

Hydroxyl amine solution (50% in water, 2.69 mL, 44.0 mmol) and 1 Maqueous sodium hydroxide solution (0.74 mL, 0.74 mmol) were added to asolution of methyl3-(1-(5-(tert-butoxycarbonylamino)pentyl)-5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(200 mg, 0.37 mmol) in THF/MeOH (4:1, 5 mL), and the resulting solutionstirred for 2 h at room temperature. The pH value of the solution wasadjusted to 2 with 6 M aqueous HCl solution and the resulting solutionstirred at room temperature for 15 h. The pH value of the solution wasadjusted to 6 with 4 M aqueous sodium hydroxide solution and the crudeproduct was purified by prep-HPLC with the following conditions: Column,XBridge Prep C18 OBD Column, 150 mm 5 um; mobile phase, water (0.1% FA)and ACN (20.0% ACN up to 30.0% in 8 min); Detector, UV 254 & 220 nm. Thecollected fraction was lyophilized to give3-(1-(5-aminopentyl)-5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideformate (3.1 mg, 2%) as an orange solid. ¹H-NMR: (DMSO, 300 MHz, ppm): δ8.50 (s, 1H), 8.27-87.98 (m, 5H), 7.46-7.38 (m, 2H), 4.41-4.35 (m, 2H),2.66-2.60 (m, 2H), 1.69-1.60 (m, 2H), 1.52-1.39 (m, 4H). MS: (ESI, m/z):447[M+H]⁺.

Example 22-1.3-(1-(5-aminopentyl)-5,6-dichloro-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideformate

Step 1: tert-butyl 5-(2-amino-4,5-dichlorophenylamino)pentylcarbamate

A solution of 4,5-dichlorobenzene-1,2-diamine (350 mg, 1.99 mmol),tert-butyl N-(5-bromopentyl)carbamate (1.05 g, 3.94 mmol), potassiumcarbonate (819 mg, 5.93 mmol), and sodium iodide (297 mg, 1.99 mmol) inDMF (20 mL) stirred for 18 h at 80° C. The resulting solution was cooledto room temperature, diluted with 50 mL of water, and extracted with3×40 mL of ethyl acetate. The combined organic phases were washed with2×30 mL of brine, dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with dichloromethane/methanol(20:1)) to give tert-butyl5-(2-amino-4,5-dichlorophenylamino)pentylcarbamate (339 mg, 44%) asbrown oil. MS: (ESI, m/z): 362[M+H]⁺.

Step 2: methyl3-(1-(5-(tert-butoxycarbonylamino)pentyl)-5,6-dichloro-1H-benzo[d]imidazol-2-ylamino)benzoate

Methyl 3-isothiocyanatobenzoate (219 mg, 1.13 mmol) was added to asolution of tert-butyl5-(2-amino-4,5-dichlorophenylamino)pentylcarbamate (339 mg, 0.87 mmol)in DMF (10 mL), and the reaction mixture stirred for 18 h at roomtemperature. DIC (164 mg, 1.30 mmol) was added, and the resultingsolution was stirred for 4 h at 80° C. The reaction mixture was cooledto room temperature, poured into 50 mL of water, and extracted with 3×50mL of ethyl acetate. The combined organic phases were washed with 3×30mL of brine, dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum. The residue was purified via columnchromatography with silica gel (eluting with ethyl acetate/petroleumether (1:2)) to give methyl3-(1-(5-(tert-butoxycarbonylamino)pentyl)-5,6-dichloro-1H-benzo[d]imidazol-2-ylamino)benzoate(300 mg, 38%) as brown oil. MS: (ESI, m/z): 521 [M+H]⁺.

Step 3:3-(1-(5-aminopentyl)-5,6-dichloro-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideformate

Hydroxyl amine solution (50% in water, 2.41 mL, 39.4 mmol) and 1 Maqueous sodium hydroxide solution (0.66 mL, 0.66 mmol) were added to asolution of methyl3-(1-(5-(tert-butoxycarbonylamino)pentyl)-5,6-dichloro-1H-benzo[d]imidazol-2-ylamino)benzoate(300 mg, 0.33 mmol) in THF/MeOH (4:1, 5 mL), and the resulting solutionstirred for 5 h at room temperature. The pH value of the solution wasadjusted to 2 with 6 M aqueous HCl solution and the resulting solutionstirred at room temperature for 15 h. The pH value of the solution wasthen adjusted to 6 with 4 M aqueous sodium hydroxide solution. Thesolids were collected by filtration and purified by prep-HPLC with thefollowing conditions: Column, XBridge Shield RP18 OBD Column, 5 um,19×150 mm; mobile phase, water (0.1% FA) and ACN (3.0% ACN up to 30.0%in 9 min); Detector, UV 220/254 nm. The collected fraction waslyophilized to give3-(1-(5-aminopentyl)-5,6-dichloro-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideformate (36.0 mg, 23%) as an off-white solid. ¹H-NMR: (DMSO, 400 MHz) δ(ppm): 8.85 (br, 3H), 8.47 (s, 1H), 8.17-8.15 (m, 2H), 7.68 (s, 1H),7.57 (s, 1H), 7.40-7.36 (m, 1H), 7.31 (d, J=7.6 Hz, 1H), 4.26-4.22 (m,2H), 2.71-2.65 (m, 2H), 1.67-1.61 (m, 2H), 1.57-1.50 (m, 2H), 1.40-1.32(m, 2H). MS: (ESI, m/z): 422[M+H]⁺.

The following compound was prepared according to the procedures outlinedabove for3-(1-(5-aminopentyl)-5,6-dichloro-1H-benzo[d]imidazol-2-ylamino)-N-hydroxybenzamideformate

(ESI, m/z) Ex. Structure Name ¹H NMR [M + H]⁺ 22-2

3-((1-(5- aminopentyl)- 5-chloro-6- fluoro-1H- benzo[d] imidazol-2-yl)amino)-N- hydroxy- benzamide formate (DMSO, 300 MHz, ppm): 8.80 (s,2H), 8.48 (s, 1H), 8.17- 8.15 (m, 2H), 7.59- 7.50 (m, 2H), 7.41- 7.29(m, 2H), 4.25- 4.21 (m, 2H), 2.69- 2.65 (m, 2H), 1.66- 1.62 (m, 2H),1.52- 1.50 (m, 2H), 1.34- 1.32 (m, 2H) 406 [M − FA + H]⁺

Example 23-1.N-hydroxy-3-((1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

Step 1: methyl3-((1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate

A solution of 2-bromo-1-(2-methoxyethyl)-1H-benzo[d]imidazole (150 mg,0.58 mmol), methyl 3-aminobenzoate (89 mg, 0.58 mmol), andmethanesulfonic acid (76 μL, 1.17 mmol) in DMA (3.9 mL) was heatedovernight at 80° C. The resulting mixture was cooled to room temperatureand diluted with ethyl acetate and water. The resulting whiteprecipitate was collected by suction filtration and dried under vacuumto give methyl3-((1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate (150 mg,78%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 11.05 (s, 1H)10.88-11.17 (m, 1H) 8.10 (s, 1H) 7.80-7.93 (m, 2H) 7.58-7.72 (m, 2H)7.39-7.43 (m, 1H) 7.25-7.37 (m, 2H) 4.58 (br t, J=4.98 Hz, 2H) 3.89 (s,3H) 3.75 (t, J=5.13 Hz, 2H) 3.27 (s, 3H). MS: (ESI, m/z): 326[M+H]⁺.

Step 2:N-hydroxy-3-((1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

Hydroxyl amine solution (50% in water, 2.5 mL, 41.5 mmol) and 1 Maqueous sodium hydroxide solution (1.38 mL, 1.38 mmol) were added to asolution of methyl3-((1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate (150 mg,0.46 mmol) in THF/MeOH (4:1, 4 mL), and the resulting solution stirredfor 2 h at room temperature. The reaction mixture was concentrated byhalf then 1 N aqueous HCl solution was added until the solution becameacidic. The resulting white precipitate was collected by suctionfiltration and dried under high vacuum to giveN-hydroxy-3-((1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(105 mg, 70%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 11.11(s, 1H) 9.79-10.08 (m, 1H) 8.95 (br d, J=1.47 Hz, 1H) 8.00-8.17 (m, 2H)7.17-7.39 (m, 4H) 6.92-7.05 (m, 2H) 4.37 (t, J=5.42 Hz, 2H) 3.59 (t,J=5.42 Hz, 2H) 3.17 (s, 3H). MS: (ESI, m/z): 327 [M+H]⁺.

The compounds in the following table were prepared according to theprocedures forN-hydroxy-3-((1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

(ESI, m/z) Ex. Structure Name ¹HNMR [M + H]⁺ 23-2

3-((1H- benzo[d] imidazol-2- yl)amino)- N-hydroxy- 1-naphtha- mide ¹HNMR (300 MHz, DMSO-d₆) δ ppm 11.27-11.38 (m, 1 H) 11.11 (br d, J = 2.35Hz, 1 H) 9.97 (br s, 1 H) 9.18-9.29 (m, 1 H) 8.51 (d, J = 2.05 Hz, 1 H)8.22 (s, 1 H) 8.01 (d, J = 8.50 Hz, 1 H) 319 7.82-7.89 (m, 1 H)7.24-7.53 (m, 4 H) 6.94-7.08 (m, 2 H) 23-3

N-hydroxy- 3-((1-(2- methoxy- ethyl)-1H- benzo[d] imidazol-2- yl)amino)-1-naphtha- mide ¹H NMR (300 MHz, DMSO-d₆) δ ppm 11.13 (br s, 1 H) 9.23-9.32 (m, 1 H) 9.11- 9.15 (m, 1 H) 8.62 (s, 1 H) 7.98-8.05 (m, 2 H) 7.86(d, J = 7.92 Hz, 1 H) 7.33-7.53 (m, 4 H) 7.01-7.12 (m, 2 H) 4.48 (br t,J = 5.28 Hz, 2 H) 3.68 (t, J = 377 5.28 Hz, 2 H) 3.25 (s, 3 H) 23-4

3-((5,6- dimethyl-1H- benzo[d] imidazol-2- yl)amino)-N- hydroxy-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ ppm 11.14 (s, 1 H) 10.74- 10.81(m, 1 H) 9.46 (br d, J = 2.05 Hz, 1 H) 9.01 (br s, 1 H) 8.14 (s, 1 H)7.98 (s, 1 H) 7.30-7.38 (m, 1 H) 7.20 (br d, J = 7.92 Hz, 297 1 H) 7.13(br s, 1 H) 7.06 (br d, J = 2.35 Hz, 1 H) 2.25 (s, 6 H)

Example 24-1.N-hydroxy-3-((4-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

Step 1: methyl 3-((4-methoxy-1H-benzo[d]imidazol-2-yl)amino)benzoate

A solution of 2-chloro-4-methoxy-1H-benzo[d]imidazole (100 mg, 0.54mmol), methyl 3-aminobenzoate (83 mg, 0.54 mmol), and methanesulfonicacid (71 μL, 1.1 mmol) in NMP (3 mL) was heated overnight at 80° C. Theresulting mixture was cooled to room temperature and then diluted withethyl acetate and water. The resulting white precipitate was collectedby suction filtration and dried under vacuum to give methyl3-((4-methoxy-1H-benzo[d]imidazol-2-yl)amino)benzoate (100 mg, 61%) as awhite solid. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 10.76 (s, 1H) 8.07 (br d,J=2.35 Hz, 1H) 7.73-7.87 (m, 2H) 7.54-7.66 (m, 1H) 7.15-7.24 (m, 1H)7.03 (d, J=7.92 Hz, 1H) 6.91 (br d, J=8.50 Hz, 1H) 3.95 (s, 3H) 3.88 (s,3H). MS: (ESI, m/z): 298[M+H]⁺.

Step 2: methyl3-((4-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate

Sodium hydride (60% dispersion in mineral oil, 13.5 mg, 0.33 mmol) wasadded to a solution of methyl3-((4-methoxy-1H-benzo[d]imidazol-2-yl)amino)benzoate (100 mg, 0.33mmol) in DMF (3 mL), and the mixture stirred for 20 minutes.1-Bromo-2-methoxyethane (0.031 mL, 0.33 mmol) was added, and theresulting solution stirred overnight. The reaction was quenched by theaddition of water (10 mL) and the resulting solution was extracted with3×10 mL of ethyl acetate. The combined organics were concentrated undervacuum, and the residue was purified via column chromatography on silicagel (gradient elution with 5-40% ethyl acetate/hexane) to give methyl3-(4-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-ylamino)benzoate(31 mg, 26%) as a colorless oil. MS: (ESI, m/z): 356 [M+H]⁺.

Step 3:N-hydroxy-3-((4-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

Hydroxyl amine solution (50% in water, 0.16 mL, 2.6 mmol) and 1 Maqueous sodium hydroxide solution (0.26 mL, 0.26 mmol) were added to asolution of methyl3-((4-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(31 mg, 0.087 mmol) in THF/MeOH (4:1, 2.5 mL), and the resultingsolution stirred for 2 h at room temperature. 2 N aqueous HCl solutionwas added until the solution was acidic, and the resulting solution wasconcentrated. The residue was purified by Gilson prep-HPLC withacetonitrile and water (0.1% formic acid) and the desired fractions areconcentrated and then lyophilized to giveN-hydroxy-3-((4-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(20 mg, 65%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 11.17 (s,1H) 9.02 (d, J=1.47 Hz, 1H) 8.87 (s, 1H) 8.02-8.13 (m, 2H) 7.30-7.41 (m,1H) 7.25 (br d, J=7.62 Hz, 1H) 7.00 (q, J=8.01 Hz, 2H) 6.69 (br d,J=7.62 Hz, 1H) 4.59 (br t, J=5.72 Hz, 2H) 3.89 (s, 3H) 3.63 (t, J=5.72Hz, 2H) 3.23 (s, 3H). MS: (ESI, m/z): 357 [M+H]⁺.

The compound in the following table was prepared according to theprocedures forN-hydroxy-3-((4-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide.

(ESI, m/z) Ex. Structure Name ¹H NMR [M + H]⁺ 24-2

N-hydroxy-3-((7- methoxy-1-(2- methoxyethyl)-1H- benzo[d]imidazol-2-yl)amino)benzamide NA 357

Example 25-1.N-hydroxy-3-((4-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

Step 1: methyl 3-((2-methoxyethyl)amino)benzoate

A solution of methyl 3-bromobenzoate (500 mg, 2.32 mmol), cesiumcarbonate (1.136 g, 3.49 mmol), Pd₂(dba)₃ (42 mg, 0.047 mmol), BINAP (43mg, 0.07 mmol), and 2-methoxyethanamine (240 μL, 1.2 mmol) in toluene(10 mL) was heated overnight at 80° C. The resulting mixture was cooledto room temperature, diluted with ethyl acetate, and washed with brine.The organic layer was separated and concentrated, and the residue waspurified via column chromatography on silica gel (gradient elution with20-70% ethyl acetate/hexane) to give methyl3-((2-methoxyethyl)amino)benzoate (250 mg, 51%) as a yellow semi-solid.¹H NMR (300 MHz, DMSO-d₆) δ ppm 7.09-7.23 (m, 3H) 6.78-6.89 (m, 1H) 5.96(t, J=5.72 Hz, 1H) 3.79-3.81 (m, 3H) 3.48 (t, J=5.72 Hz, 2H) 3.27 (s,3H) 3.15-3.25 (m, 2H). MS: (ESI, m/z): 210[M+H]⁺.

Step 2: methyl3-((1H-benzo[d]imidazol-2-yl)(2-methoxyethyl)amino)benzoate

A solution of 2-bromo-1H-benzo[d]imidazole (57 mg, 0.28 mmol), methyl3-(2-methoxyethylamino)benzoate (60 mg, 0.28 mmol), and 6 M aqueous HClsolution (1 drop) in ethanol (1.5 mL) was irradiated with microwaveradiation for 1 h at 150° C. The resulting mixture was cooled to roomtemperature and then concentrated under vacuum. The residue was purifiedvia prep-HPLC with acetonitrile and water (0.1% formic acid) and thedesired fractions were concentrated and then lyophilized to give methyl3-((1H-benzo[d]imidazol-2-yl)(2-methoxyethyl)amino)benzoate (35 mg, 38%)as a white solid. MS: (ESI, m/z): 326[M+H]⁺.

Step 3:3-((1H-benzo[d]imidazol-2-yl)(2-methoxyethyl)amino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.33 mL, 5.4 mmol) and 1 Maqueous sodium hydroxide solution (0.33 mL, 0.33 mmol) were added to asolution of methyl3-((1H-benzo[d]imidazol-2-yl)(2-methoxyethyl)amino)benzoate (35 mg,0.108 mmol) in THF/MeOH (4:1, 2.5 mL), and the resulting solutionstirred for 2 h at room temperature. 2 N aqueous HCl solution was addeduntil the solution was acidic, and the resulting solution wasconcentrated. The residue was purified by Gilson prep-HPLC withacetonitrile and water (0.1% formic acid) and the desired fractions wereconcentrated and then lyophilized to give3-((1H-benzo[d]imidazol-2-yl)(2-methoxyethyl)amino)-N-hydroxybenzamide(7 mg, 20%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 11.27 (brs, 2H) 8.98-9.20 (m, 1H) 8.16 (s, 1H) 7.76 (s, 1H) 7.61 (br d, J=6.74Hz, 1H) 7.50-7.58 (m, 1H) 7.31-7.50 (m, 1H) 7.21-7.31 (m, 1H) 7.12 (brd, J=3.22 Hz, 1H) 6.93 (s, 1H) 6.93 (br dd, J=11.29, 4.84 Hz, 2H) 4.09(br d, J=6.16 Hz, 2H) 3.55-3.64 (m, 2H) 3.23 (s, 3H). MS: (ESI, m/z):327 [M+H]⁺.

Example 26-1:3-(5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yloxy)-N-hydroxybenzamide

Step 1:2-mercapto-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile

Carbon disulfide (4.80 mL, 79.6 mmol) was added in portions to asolution of 4,5-diamino-2-(trifluoromethyl)benzonitrile (2.00 g, 9.94mmol) and potassium hydroxide (1.67 g, 29.76 mmol) in ethanol (100 mL),and the resulting solution stirred for 5 h at 90° C. in an oil bath. Theresulting mixture was cooled to room temperature and then concentratedunder vacuum. The residue was diluted with 100 mL of water, the pH valueof the resulting solution was adjusted to 7 with 2 M aqueous HClsolution, and the mixture was extracted with 3×200 mL of ethyl acetate.The combined organic phases were washed with 1×100 mL of brine, driedover anhydrous sodium sulfate, filtered, and concentrated under vacuumto afford2-mercapto-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile (1.9g, 79%) as a red solid. MS: (ESI, m/z): 244[M+H]⁺.

Step 2: 2-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile

Bromine (0.20 mL, 3.96 mmol) was added dropwise to a 0° C. solution of2-mercapto-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile (267mg, 1.10 mmol) in hydrobromic acid (40% in acetic acid, 12 mL), and theresulting solution stirred for 3 h at 0° C. in a water/ice bath. Thereaction mixture was concentrated under vacuum, and the residue wasdiluted with 10 mL of water. The pH value of the solution was adjustedto 4 with 1 M aqueous sodium hydroxide solution. The resulting solutionwas extracted with 3×20 mL of ethyl acetate, and the combined organicphases were dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum. The residue was purified via columnchromatography on silica gel (eluting with ethyl acetate/petroleum ether(1:1)) to afford2-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile (225 mg,71%) as a yellow solid. MS: (ESI, m/z): 331 [M+H+CH₃CN]⁺.

Step 3: methyl3-(5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yloxy)benzoate

A solution of2-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazole-5-carbonitrile (200 mg,0.69 mmol), methyl 3-hydroxybenzoate (105 mg, 0.69 mmol), copper (5 mg),and potassium carbonate (286 mg, 2.05 mmol) in pyridine (20 mL) stirredfor 24 h at 110° C. in an oil bath. The resulting solution was cooled toroom temperature and then concentrated under vacuum. The residue wasdiluted with 20 mL of water and extracted with 3×20 mL of ethyl acetate.The combined organic phases were washed with 50 mL of water and 50 mL ofbrine, dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography onsilica gel (eluting with methanol/dichloromethane (1:25)) to affordmethyl3-(5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yloxy)benzoate (95mg, 38%) as a yellow solid. MS: (ESI, m/z): 362[M+H]⁺

Step 4:3-(5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yloxy)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.27 mL, 4.2 mmol) and 1 Maqueous sodium hydroxide solution (0.28 mL, 0.28 mmol) were added to asolution of methyl3-(5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yloxy)benzoate (50mg, 0.14 mmol) in THF/MeOH (4:1, 1.5 mL), and the resulting solutionstirred for 6 h at room temperature. The crude product was purified byPrep-HPLC with the following conditions: Column, XBridge RP C18, 19×150mm, 5 um; mobile phase, Mobile Phase A: water/0.05% TFA, Mobile Phase B:ACN; Flow rate: 25 mL/min; Gradient: 5% B to 54% B in 7.0 min; Detector,254 nm. The collected fraction was lyophilized to afford3-(5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yloxy)-N-hydroxybenzamide(4.6 mg, 9%) as a red solid. ¹H-NMR: (DMSO, 300 MHz) δ (ppm): 11.31 (s,1H), 9.13 (s, 1H), 8.18 (s, 1H), 7.94 (s, 1H), 7.76-7.67 (m, 2H),7.59-7.57 (m, 2H). MS: (ESI, m/z): 363 [M+H]⁺

Example 27-1:3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-N-hydroxybenzamide

Step 1: methyl3-(2-(2-amino-5-cyano-4-(trifluoromethyl)phenylamino)-2-oxoethyl)benzoate

A solution of 4,5-diamino-2-(trifluoromethyl)benzonitrile (100 mg, 0.50mmol), DMC (96 mg, 0.57 mmol), 2-[3-(methoxycarbonyl)phenyl]acetic acid(91.9 mg, 0.47 mmol), and N,N-diisopropylethyl amine (0.30 mL, 1.9 mmol)in dichloromethane (10 mL) stirred for 1 h at 25° C. The reactionmixture was then poured into 10 mL of water. The resulting solution wasextracted with 3×10 mL of ethyl acetate, and the combined organic phaseswere dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography onsilica gel (eluting with ethyl acetate/petroleum ether (1:4)) to affordmethyl3-(2-(2-amino-5-cyano-4-(trifluoromethyl)phenylamino)-2-oxoethyl)benzoate(160 mg, 90%) as a white solid. MS: (ESI, m/z): 378[M+H]⁺.

Step 2: methyl3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)benzoate

A solution of methyl3-(2-(2-amino-5-cyano-4-(trifluoromethyl)phenylamino)-2-oxoethyl)benzoate(100 mg, 0.27 mmol) in toluene (10 mL) and acetic acid (1 mL) stirredfor 2 h at 110° C. in an oil bath. The resulting mixture was cooled toroom temperature and then concentrated under vacuum. The residue waspurified via column chromatography on silica gel (eluting with ethylacetate/petroleum ether (1:3)) to afford methyl3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)benzoate(65 mg, 68%) as a yellow solid. MS: (ESI, m/z): 360[M+H]⁺.

Step 3:3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.52 mL, 7.9 mmol) and 1 Maqueous sodium hydroxide solution (0.53 mL, 0.53 mmol) were added to asolution of methyl3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)benzoate(95 mg, 0.26 mmol) in THF/MeOH (4:1, 2.0 mL), and the resulting solutionstirred for 1.5 h at room temperature. The crude product was purified byPrep-HPLC with the following conditions: Column: Waters XBridge C18,19*150 mm; Mobile Phase A: water/0.05% TFA, Mobile Phase B: ACN; Flowrate: 25 mL/min; Gradient: 30% B to 70% B in 10 min; Detector: 254 nm.The collected fraction was lyophilized to afford3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-N-hydroxybenzamide(34.3 mg, 36%) as a pink solid. ¹H-NMR: (DMSO, 400 MHz) δ (ppm): 11.20(br, 1H), 8.38 (s, 1H), 8.12 (s, 1H), 7.74 (s, 1H), 7.62 (d, J=7.6 Hz,1H), 7.50 (d, J=7.6 Hz, 1H), 7.43-7.39 (m, 1H), 4.36 (s, 2H). MS: (ESI,m/z): 361[M+H]⁺.

The following compound was prepared according to the procedures outlinedabove3-((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-N-hydroxybenzamide.

(ESI, m/z) Ex. Structure Name ¹H NMR [M + H]⁺ 27-2

3-((5,6-dichloro- 1H-benzo[d]imidazol- 2-yl)methyl)-N- hydroxybenzamide(DMSO, 300 MHz, ppm): 7.89 (s, 2H), 7.75 (s, 1H), 7.63 (d, J = 7.5 Hz,1H), 7.51-7.39 (m, 2H), 4.32 (s, 2H) 336

Example 28-1: 3-(5,6-dichloro-1H-indol-2-ylamino)-N-hydroxybenzamide

Step 1: 1,2-dichloro-4-methyl-5-nitrobenzene

Fuming nitric acid (40 mL) was added dropwise to 0° C. solution of1,2-dichloro-4-methylbenzene (20 g, 124.20 mmol), and the resultingsolution was stirred for 2 h at room temperature. The reaction mixturewas then slowly poured into 200 mL of water/ice. The solids werecollected by filtration and dried under vacuum to afford1,2-dichloro-4-methyl-5-nitrobenzene (25.2 g, 98%) as a yellow solid.¹H-NMR: (DMSO, 300 MHz) δ (ppm): 8.39 (s, 1H), 7.96 (s, 1H), 2.48 (s,3H).

Step 2: (E)-2-(4,5-dichloro-2-nitrophenyl)-N,N-dimethylethenamine

A solution of 1,2-dichloro-4-methyl-5-nitrobenzene (25.2 g, 122 mmol),DMF (50 mL), and DMF-dimethyl acetal (19.8 mL, 147.90 mmol) was stirredfor 3 h at 130° C. The resulting mixture was concentrated under vacuum.The residue was dissolved in 200 mL of ethyl acetate, washed with 200 mLof water and 200 mL of brine, dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum to afford(E)-2-(4,5-dichloro-2-nitrophenyl)-N,N-dimethylethenamine (37 g) as ared solid. MS: (ESI, m/z): 261[M+H]⁺.

Step 3: 5,6-dichloro-1H-indole

Iron filings (133 g, 2.39 mmol) were added to a 60° C. solution of(E)-2-(4,5-dichloro-2-nitrophenyl)-N,N-dimethylethenamine (37 g, 120mmol) and acetic acid (500 mL) in ethanol (500 mL), and the resultingsolution stirred for 3 h at 90° C. The reaction mixture was cooled toroom temperature and filtered. The filtrate was concentrated, and theresidue was diluted with 500 mL of water and extracted with 2×500 mL ofethyl acetate. The combined organic phases were washed with 500 mL ofbrine, dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography onsilica gel (eluting with ethyl acetate/petroleum ether (1:10)) to afford5,6-dichloro-1H-indole (6 g, 27%) as a brown solid. MS: (ESI, m/z):186[M+H]⁺.

Step 4: tert-butyl 5,6-dichloro-1H-indole-1-carboxylate

A solution of 5,6-dichloro-1H-indole (1.7 g, 9.14 mmol),4-dimethylaminopyridine (1.4 g, 11.46 mmol), di-tert-butyl dicarbonate(2.4 g, 11.00 mmol), and THF (50 mL) stirred overnight at roomtemperature. The mixture was then diluted with 50 mL of ethyl acetate,washed with 50 mL of brine, dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum. The residue was purified viacolumn chromatography on silica gel (eluting with ethylacetate/petroleum ether (1:30)) to afford tert-butyl5,6-dichloro-1H-indole-1-carboxylate (2.0 g, 76%) as a light yellowsolid. MS: (ESI, m/z): 286[M+H]⁺.

Step 5: tert-butyl 5,6-dichloro-2-iodo-H-indole-1-carboxylate

t-Butyllithium (1.5 M in pentane, 3.0 mL, 4.5 mmol) was added dropwiseto a −78° C. solution of tert-butyl 5,6-dichloro-1H-indole-1-carboxylate(1.25 g, 4.37 mmol) in THF (50 mL), and the mixture stirred for 1 h at−78° C. A solution of iodine (3.3 g, 12.99 mmol) in THF (5 mL) was addeddropwise, and the resulting solution stirred for 2 h at −78° C. Thereaction was quenched by the addition of 5 mL of saturated aqueousammonium chloride solution and then poured into 100 mL of water. Themixture was extracted with 3×100 mL of ethyl acetate, and the combinedorganic phases were washed with 100 mL of brine, dried over anhydroussodium sulfate, filtered, and concentrated under vacuum. The residue waspurified via column chromatography on silica gel (eluting with ethylacetate/petroleum ether (1:20)) to afford tert-butyl5,6-dichloro-2-iodo-1H-indole-1-carboxylate (1 g, 56%) as a pink solid.MS: (ESI, m/z): 412[M+H]⁺.

Step 6: tert-butyl5,6-dichloro-2-(3-(methoxycarbonyl)phenylamino)-1H-indole-1-carboxylate

A solution of tert-butyl 5,6-dichloro-2-iodo-1H-indole-1-carboxylate(120 mg, 0.29 mmol), methyl 3-aminobenzoate (60 mg, 0.40 mmol), cesiumcarbonate (180 mg, 0.55 mmol), Pd₂(dba)₃ (5 mg) and Xantphos (5 mg, 0.01mmol) in toluene (12 mL) stirred for 1 h at 100° C. and was then cooledto room temperature. The mixture was concentrated under vacuum, and theresidue was diluted with 20 mL of water and extracted with 2×20 mL ofethyl acetate. The combined organic phases were washed with 50 mL ofbrine, dried over anhydrous sodium sulfate, filtered, and concentratedunder vacuum. The residue was purified via column chromatography onsilica gel (eluting with ethyl acetate/petroleum ether (1:1)) to affordtert-butyl5,6-dichloro-2-(3-(methoxycarbonyl)phenylamino)-1H-indole-1-carboxylate(70 mg, 55%) as red oil. MS: (ESI, m/z): 335 [M-Boc+H]⁺.

Step 7: 3-(5,6-dichloro-1H-indol-2-ylamino)-N-hydroxybenzamide(PH-FMA-HD-400-0)

Hydroxyl amine solution (50% in water, 0.55 mL, 8.3 mmol) and 1 Maqueous sodium hydroxide solution (0.28 mL, 0.28 mmol) were added to asolution of tert-butyl5,6-dichloro-2-(3-(methoxycarbonyl)phenylamino)-1H-indole-1-carboxylate(60 mg, 0.14 mmol) in THF/MeOH (4:1, 2.0 mL), and the resulting solutionstirred for 4 h at room temperature. The crude product was purified viaprep-HPLC with the following conditions: Column: XBridge RP C18, 19×150mm, 5 um; Mobile Phase A: water/0.05% TFA, Mobile Phase B: ACN; Flowrate: 30 mL/min; Gradient: 5% B to 40% B in 6.0 min, 254 nm. Thecollected fraction was lyophilized to afford3-(5,6-dichloro-1H-indol-2-ylamino)-N-hydroxybenzamide (16.6 mg, 36%) asa gray solid. ¹H-NMR: (DMSO, 400 MHz) δ (ppm): 11.17 (br, 1H), 10.87 (s,1H), 8.93 (s, 1H), 7.61 (s, 2H), 7.40 (s, 1H), 7.35-7.27 (m, 1H),7.25-7.19 (m, 2H), 5.98 (s, 1H). MS: (ESI, m/z): 336 [M+H]⁺.

Example 29-1: 3-(1H-imidazo[4,5-b]pyrazin-2-ylamino)-N-hydroxybenzamide

Step 1: methyl 3-(1H-imidazo[4,5-b]pyrazin-2-ylamino)benzoate

A solution of pyrazine-2,3-diamine (540 mg, 4.91 mmol) and methyl3-isothiocyanatobenzoate (1.04 g, 5.40 mmol) in DMF (10 mL) stirred for2 h at 100° C. in an oil bath. The resulting mixture was cooled to roomtemperature and then concentrated under vacuum. The residue was purifiedvia column chromatography on silica gel (eluting withmethanol/dichloromethane (1:5)) to afford methyl3-(1H-imidazo[4,5-b]pyrazin-2-ylamino)benzoate (60 mg, 5%) as a yellowsolid. MS: (ESI, m/z): 270 [M+H]⁺.

Step 2: 3-(1H-imidazo[4,5-b]pyrazin-2-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.15 mL, 2.3 mmol) and 1 Maqueous sodium hydroxide solution (0.23 mL, 0.23 mmol) were added to asolution of methyl 3-(1H-imidazo[4,5-b]pyrazin-2-ylamino)benzoate (21mg, 0.08 mmol) in THF/MeOH (4:1, 2.0 mL), and the resulting solutionstirred for 30 minutes at room temperature. The pH value of the solutionwas adjusted to 5-6 with 1 M aqueous HCl solution. The crude product waspurified via Prep-HPLC with conditions: Column: XBridge RP C18, 19×150mm, 5 um; Mobile Phase A: water/0.05% TFA, Mobile Phase B: ACN; Flowrate: 25 mL/min; Gradient: 4% B to 20% B in 7.0 min, 254 nm. Thecollected fraction was lyophilized to afford3-(1H-imidazo[4,5-b]pyrazin-2-ylamino)-N-hydroxybenzamide (16.7 mg, 79%)as a yellow solid. ¹H-NMR: (DMSO, 300 MHz) δ (ppm): 11.19 (br, 1H),10.26 (br, 1H), 8.16 (s, 1H), 8.02 (s, 2H), 7.96 (d, J=7.5 Hz, 1H),7.46-7.37 (m, 2H). MS: (ESI, m/z): 271 [M+H]⁺.

Example 30-1: 3-(9H-purin-8-ylamino)-N-hydroxybenzamide

Step 1: methyl 3-(9H-purin-8-ylamino)benzoate

A solution of methyl 3-isothiocyanatobenzoate (170 mg, 0.88 mmol) in THF(2 mL) was added dropwise to a solution of pyrimidine-4,5-diamine (100mg, 0.91 mmol) in THF (8 mL), and the resulting solution stirredovernight at 65° C. in an oil bath. DIC (0.43 mL, 2.72 mmol) was thendropwise, and the resulting solution was stirred for 5 h at 65° C. in anoil bath. The reaction mixture was cooled to room temperature and thendiluted with 25 mL of water. The mixture was extracted with 3×20 ofethyl acetate, and the combined organic phases were washed with 1×10 mLof brine, dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum. The residue was purified via preparative thinlayer chromatography (eluting with methanol/dichloromethane (1:10)) toafford methyl 3-(9H-purin-8-ylamino)benzoate (43 mg, 18%) as a lightyellow solid. MS: (ESI, m/z): 270 [M+H]⁺.

Step 2: 3-(9H-purin-8-ylamino)-N-hydroxybenzamide

Hydroxyl amine solution (50% in water, 0.26 mL, 3.9 mmol) and 1 Maqueous sodium hydroxide solution (0.39 mL, 0.39 mmol) were added to asolution of methyl 3-(9H-purin-8-ylamino)benzoate in THF/MeOH (4:1, 2.0mL), and the resulting solution stirred for 30 minutes at roomtemperature. The pH value of the solution was adjusted to 5-6 with 1 Maqueous HCl solution. The crude product was purified by Prep-HPLC withconditions: Column: XBridge RP C18, 19×150 mm, 5 um; Mobile Phase A:water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 4%B to 10% B in 7.0 min, 254 nm. The collected fraction was lyophilized toafford 3-(9H-purin-8-ylamino)-N-hydroxybenzamide (8.9 mg, 26%) as alight brown solid. ¹H-NMR: (DMSO, 400 MHz) δ (ppm): 11.21 (br, 1H),10.72 (br, 1H), 8.84 (s, 1H), 8.64 (s, 1H), 8.12 (s, 1H), 7.94 (d, J=7.6Hz, 1H), 7.50-7.41 9m, 2H). MS: (ESI, m/z): 271 [M+H]⁺.

Example 31-1:6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-N-hydroxypicolinamide

Step 1: methyl 6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)picolinate

A solution of Pd(OAc)₂ (3 mg, 0.01 mmol) and t-BuBrettPhos (12 mg, 0.02mmol) in tert-butanol (1 mL) and water (1 drop) stirred for 1.5 min at110° C. in an oil bath and was then added dropwise via cannula to asolution of 5,6-dichloro-1,3-benzoxazol-2-amine (50 mg, 0.25 mmol),methyl 6-bromopyridine-2-carboxylate (53 mg, 0.25 mmol), and potassiumcarbonate (47.6 mg, 0.34 mmol) in tert-butanol (2 mL). The resultingsolution stirred for 3 h at 110° C. and was then cooled to roomtemperature and concentrated under vacuum. The residue was diluted with10 mL of water and extracted with 3×10 mL of ethyl acetate. The combinedorganic phases were dried over anhydrous sodium sulfate, filtered, andconcentrated under vacuum to afford methyl6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)picolinate (95 mg) as a yellowsolid. MS: (ESI, m/z): 338[M+H]⁺.

Step 2: 6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)picolinic acid

A solution of lithium hydroxide (71 mg, 2.97 mmol) in water (2 mL) wasadded to a solution of methyl6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)picolinate (110 mg, 0.30 mmol)in THF (2 mL), and the resulting solution stirred for 2 h at roomtemperature. THF was removed under vacuum and the reaction mixture wasdiluted with 5 mL of water. The resulting solution was washed with 5 mLof ethyl acetate and the aqueous layer was separated and cooled to 0° C.The pH value of the solution was adjusted to 5 with 2 M aqueous HClsolution, and the resulting solution was extracted with 3×10 mL of ethylacetate. The combined organic phases were dried over anhydrous sodiumsulfate, filtered, and concentrated under vacuum to afford6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)picolinic acid (65 mg) as ayellow solid. MS: (ESI, m/z): 324[M+H]⁺.

Step 3: 6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-N-hydroxypicolinamide

IPCF (20 μL, 0.18 mmol) was added dropwise to a 0° C. solution of6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)picolinic acid (65 mg, 0.18mmol) and NMM (99 μL, 0.91 mmol) in DMA (5 mL). Hydroxylaminehydrochloride (14 mg, 0.20 mmol) was added, and the resulting solutionstirred overnight at room temperature. The solids were filtered out. Thecrude product was purified by Prep-HPLC with the following conditions:Column: Waters HSS C18, 2.1×50 mm, 1.8 um; Mobile Phase A: water/0.05%TFA, Mobile Phase B: ACN; Flow rate: 0.7 mL/min; Gradient: 5% B to 95% Bin 2.0 min, hold 0.6 min; Detector: UV 254 nm. The collected fractionwas lyophilized to afford6-(5,6-dichlorobenzo[d]oxazol-2-ylamino)-N-hydroxypicolinamide (6.4 mg,11%) as a white solid. ¹H-NMR: (DMSO, 300 MHz) δ (ppm): 11.59 (s, 1H),10.73 (s, 1H), 9.27 (s, 1H), 8.29 (d, J=8.1 Hz, 1H), 8.08-8.00 (m, 2H),7.80 (s, 1H), 7.60 (d, J=7.5 Hz, 1H). MS: (ESI, m/z): 339[M+H]⁺.

Example 32-1: Preparation ofN-hydroxy-3-(oxazolo[4,5-b]pyridin-2-ylamino)benzamide

Step 1: methyl 3-(oxazolo[4,5-b]pyridin-2-ylamino)benzoate

A solution of 2-aminopyridin-3-ol (440 mg, 4.00 mmol), methyl3-isothiocyanatobenzoate (0.63 mL, 4.00 mmol), copper (I) iodide (38 mg,0.20 mmol), and triethylamine (2.22 mL, 16.0 mmol) in ethanol (8 mL) wasirradiated with microwave radiation for 10 min at 100° C. The resultingmixture was cooled to room temperature and then concentrated undervacuum. The residue was purified via Prep-HPLC with the followingconditions: Column, XBridge RP18 19*250 mm; Mobile phase A: water (0.1%FA), Mobile Phase B: ACN; Flow rate: 30 mL/min; Gradient: 20% B to 80% Bin 10 min; 254&220 nm. The collected fraction was concentrated undervacuum to afford methyl 3-(oxazolo[4,5-b]pyridin-2-ylamino)benzoate (150mg, 14%) as a light yellow solid. MS: (ESI, m/z): 270[M+H]⁺.

Step 2: 3-(oxazolo[4,5-b]pyridin-2-ylamino)benzoic acid

A solution of lithium hydroxide (22 mg, 0.92 mmol) in water (1 mL) wasadded to a solution of methyl3-(oxazolo[4,5-b]pyridin-2-ylamino)benzoate (50 mg, 0.19 mmol) in THF (1mL), and the resulting solution stirred for 12 h at room temperature.The pH value of the solution was adjusted to 7 with 6 M aqueous HClsolution, and the mixture was extracted with 3×10 mL of ethyl acetate.The combined organic phases were dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum to afford3-(oxazolo[4,5-b]pyridin-2-ylamino)benzoic acid (35 mg, 74%) as yellowoil. MS: (ESI, m/z): 256[M+H]⁺.

Step 3: N-hydroxy-3-(oxazolo[4,5-b]pyridin-2-ylamino)benzamide

IPCF (76 μL, 0.68 mmol) was added dropwise to a 0° C. solution of3-(oxazolo[4,5-b]pyridin-2-ylamino)benzoic acid (35 mg, 0.14 mmol) andNMM (75 μL, 0.91 mmol) in DMA (5 mL). The mixture stirred for 1 h atroom temperature and then a solution of hydroxylamine hydrochloride (47mg, 0.68 mmol) in DMA (1 mL) was added. The resulting solution wasstirred for 14 h at room temperature. The solids were filtered out. Thecrude product was purified by Prep-HPLC with the following conditions:Column: XBridge BEH C18 OBD Prep Column, 5 um, 19×250 mm; Mobile PhaseA: water (0.1% FA), Mobile Phase B: ACN; Flow rate: 30 mL/min; Gradient:5% B to 30% B in 6 min; 254 & 220 nm. The collected fraction waslyophilized to affordN-hydroxy-3-(oxazolo[4,5-b]pyridin-2-ylamino)benzamide (10.6 mg, 29%) asa white solid. ¹H-NMR (DMSO, 400 MHz) δ(ppm): 11.16 (br, 2H), 9.08 (br,1H), 8.27-8.25 (m, 1H), 8.17 (s, 1H), 7.94-7.87 (m, 2H), 7.50-7.41 (m,2H), 7.17-7.13 (m, 1H). MS: (ESI, m/z): 271[M+H]⁺.

The following compound was prepared according to the procedures outlinedN-hydroxy-3-(oxazolo[4,5-b]pyridin-2-ylamino)benzamide.

(ESI, m/z) Ex. Structure Name ¹HNMR [M + H]⁺ 32-2

N-hydroxy-3-((6- (trifluoromethyl)oxa- zolo[4,5-b]pyridin- 2-yl)amino)benzamide (DMSO, 300 MHz, ppm): 11.33 (br, 2H), 9.08 (br, 1H),8.60 (s, 1H), 8.30 (s, 1H), 8.14 (s, 1H), 7.92-7.85 (m, 1H), 7.51-7.39(m, 2H) 339

Example 33-1.3-((6-chloro-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide

A 2 mL reaction vial was charged with4-chloro-5-fluorobenzene-1,2-diamine (0.2 M in DMF, 150 μL, 30 umol),triethylamine (42 μL, 300 μmol) and methyl 3-isothiocyanatobenzoate (0.2M in DMF, 150 μL, 30 μmol).1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.4 M in DMF, 225 μL, 90μmol) was added, and the resulting mixture was shaken at roomtemperature for 30 minutes and then at 90° C. overnight. The solvent wasremoved under a stream of nitrogen. The residue was diluted with ethylacetate (0.7 mL) and brine (0.5 mL), and the mixture was shaken. Theorganic layer was separated and the aqueous phase was extracted withethyl acetate (0.7 mL). The combined organic layers were concentratedunder a stream of nitrogen and THF/methanol (3:1, 200 μL) was added tothe residue. The vial was sealed and shaken at 50° C. for 15 min todissolve the residue and then cooled to room temperature. Hydroxylamine(50% v/v solution in water, 150 μL) was added followed by 1 N aqueousNaOH solution (100 μL), and the vial was sealed and shaken at roomtemperature for 16 h. The reaction mixture was concentrated under astream of nitrogen at room temperature. The residue was dissolved in 500μL of DMSO and purified by prep-HPLC with the following conditions:Column: Waters SunFire C18, 5 um, 19×150 mm; mobile phase, A: water with0.1% formic acid, B: CH₃CN with 0.1% formic acid; Flow rate, 23 mL/min;Gradient, 0% B to 5% B in 5 min; Detector, 254, 220 nm. The collectedfractions were concentrated to give3-((6-chloro-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide(1.5 mg, 15.6% yield) as an off-white solid. MS: (ESI, m/z): 321[M+H]⁺.

The following compounds were prepared according to the proceduresoutlined3-((6-chloro-5-fluoro-1H-benzo[d]imidazol-2-yl)amino)-N-hydroxybenzamide:

(ESI, m/z) Example Structure Name [M + H]+ 33-2

3-((5-chloro-6-methyl- 1H-benzo[d]imidazol- 2-yl)amino)-N-hydroxybenzamide 317 33-3

3-((5,6-difluoro-1H- benzo[d]imidazol-2- yl)amino)-N- hydroxybenzamide305 33-4

3-((5H- [1,3]dioxolo[4′,5′:4,5] benzo[1,2-d]imidazol- 6- yl)amino)-N-hydroxybenzamide 313 33-5

3-((5-fluoro-6- (trifluoromethyl)-1H- benzo[d]imidazol-2- yl)amino)-N-hydroxybenzamide 355 33-6

3-((5-chloro-6- (trifluoromethyl)-1H- benzo[d]imidazol-2- yl)amino)-N-hydroxybenzamide 371 33-7

3-((5-bromo-6- (trifluoromethoxy)- 1H-benzo[d]imidazol- 2-yl)amino)-N-hydroxybenzamide 431 33-8

3-((7,8-dihydro- 1H,6H- [1,4]dioxepino[2′,3′:4, 5]benzo[1,2-d]imidazol-2- yl)amino)-N- hydroxybenzamide 341 33-9

3-((6,7-dihydro-1H- [1,4]dioxino[2′,3′:4,5] benzo[1,2-d]imidazol-2-yl)amino)-N- hydroxybenzamide 327 33-10

3-((5-fluoro-6-methyl- 1H-benzo[d]imidazol- 2-yl)amino)-N-hydroxybenzamide 301 33-11

3-((5-bromo-6- (trifluoromethyl)-1H- benzo[d]imidazol-2- yl)amino)-N-hydroxybenzamide 415 33-12

3-((5-bromo-6-methyl- 1H-benzo[d]imidazol- 2-yl)amino)-N-hydroxybenzamide 361 33-13

3-((5,6-dimethoxy-1H- benzo[d]imidazol-2- yl)amino)-N- hydroxybenzamide329 33-14

3-((5,6-dichloro-1H- benzo[d]imidazol-2- yl)amino)-N- hydroxybenzamide337 33-15

3-((5-chloro-6-nitro- 1H-benzo[d]imidazol- 2-yl)amino)-N-hydroxybenzamide 348 33-16

3-((6-bromo-5-fluoro- 1H-benzo[d]imidazol- 2-yl)amino)-N-hydroxybenzamide 365 33-17

3-((5-cyano-6- (trifluoromethyl)-1H- benzo[d]imidazol-2- yl)amino)-N-hydroxybenzamide 355 33-18

N-hydroxy-3-((7-oxo- 3,6,7,8- tetrahydroimidazo [4′,5′:4,5]benzo[1,2-b][1,4]oxazin-2- yl)amino)benzamide 340 33-19

3-((6-chloro-5- methoxy-1H- benzo[d]imidazol-2- yl)amino)-N-hydroxybenzamide 333 33-20

3-((5-chloro-1H- benzo[d]imidazol-2- yl)amino)-N- hydroxybenzamide 30333-21

N-hydroxy-3-((5-nitro- 1H-benzo[d]imidazol- 2-yl)amino)benzamide 31433-22

N-hydroxy-3-((5- (trifluoromethyl)-1H- benzo[d]imidazol-2-yl)amino)benzamide 337 33-23

N-hydroxy-3-((5- (methylsulfonyl)-1H- benzo[d]imidazol-2-yl)amino)benzamide 347 33-24

N-hydroxy-3-((5- sulfamoyl-1H- benzo[d]imidazol-2- yl)amino)benzamide348

Example 34-1.N-hydroxy-3-(methyl(1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

Step 1: Methyl3-((5-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate

A solution of 4-bromo-5-(trifluoromethyl)benzene-1,2-diamine (700 mg,2.74 mmol), triethylamine (1.4 mL, 10 mmol) and methyl3-isothiocyanatobenzoate (530 mg, 2.74 mmol) in DMF (8 mL) stirred atroom temperature for 15 minutes and then1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.063 g, 6.85 mmol) wasadded. The resulting mixture was stirred at room temperature for 30 minthen at 90° C. for 4 h. The solvent was evaporated under reducepressure, and the residue was diluted with ethyl acetate (10 mL) andbrine (5 mL). The aqueous phase was separated and extracted with ethylacetate (10 mL). The combined organic layers were dried over anhydroussodium sulfate, filtered, and concentrated under reduce pressure. Theresidue was purified via column chromatography on silica gel (elutingwith ethyl acetate/hexane (2:1)) to afford methyl3-((5-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(706 mg, 62%) as white solid. MS: (ESI, m/z): 415[M+H]⁺.

Step 2: Methyl3-((5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate

Potassium phosphate tribasic (2 M in water, 200 μL, 400 μmol) andPd(PPh₃)₄ (11.6 mg, 10 μmol) were added to a solution of methyl3-((5-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(41.4 mg, 100 μmol), and3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (51.2 mg, 250μmol) in 1,4-dioxane (600 μL) and water (60 μL) and stirred at 120° C.overnight. The mixture was cooled to room temperature and ethyl acetate(1 mL) and brine (500 μL) were added. The aqueous phase was separatedand extracted with ethyl acetate (1 mL). The combined organic layerswere dried and concentrated. The crude product was purified bypreparative thin layer chromatography (eluting withDCM/MeOH/NH₄OH=10:1:0.1) to give methyl3-((5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(29 mg, 72%) as a light yellow solid. MS: (ESI, m/z): 413 [M+H]⁺).

Step 3: Methyl3-(methyl(1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoateand methyl3-(methyl(1-methyl-6-(pyridin-3-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate

A 2 mL reaction vial was charged with methyl3-((5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(29 mg, 72 μmol) and acetonitrile (400 μL). Iodomethane (0.4 M inacetonitrile, 500 μL, 200 μmol) was added followed by cesium carbonate(130 mg, 400 μmol). The mixture was shaken at room temperatureovernight, and the solvent was removed under reduced pressure. Theresidue was diluted with ethyl acetate (0.7 mL) and brine (0.5 mL), andthe mixture was shaken. The aqueous phase was separated and extractedwith ethyl acetate (0.7 mL), and the combined organic layers wereconcentrated under a stream of nitrogen. The crude product was purifiedby preparative thin layer chromatography (eluting with 10:0.5:0.05DCM/MeOH/NH₄OH). The top band on the TLC plate was assumed as methyl3-(methyl(1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(9.5 mg, 31%) MS: (ESI, m/z): 441[M+H]⁺. The lower band was assumed asmethyl3-(methyl(1-methyl-6-(pyridin-3-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(3.7 mg, 12%) MS: (ESI, m/z): 441[M+H]⁺.

Step 4:(N-hydroxy-3-(methyl(1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide)

A solution of methyl3-(methyl(1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(9.5 mg, 22 μmol) in THF/methanol (3:1, 200 μL) was heated at 50° C. for15 minutes (to dissolve the solids) and then cooled to room temperature.Hydroxylamine (50% v/v solution in water, 150 μL) and 1 N aqueous NaOHsolution (100 μL) were added, and the mixture was shaken at roomtemperature for 16 h. The reaction mixtures was concentrated under astream of nitrogen at room temperature. The residue was dissolved in 500μL of DMSO and purified by prep-HPLC with the following conditions:Column: Waters SunFire C18, 5 um, 19×150 mm; mobile phase, A: water with0.1% formic acid, B: CH₃CN with 0.1% formic acid; Flow rate, 23 mL/min;Gradient, 0% B to 5% B in 5 min; Detector, 254, 220 nm. The collectedfractions were concentrated to give(N-hydroxy-3-(methyl(1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide)(5.8 mg, 61% yield), MS: (ESI, m/z): 442[M+H]⁺

The following compounds were prepared according to the proceduresoutlinedN-hydroxy-3-(methyl(1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide:

(ESI, m/z) Example Structure IUPAC Name [M + H]+ 34-2

(N-hydroxy-3- (methyl(1-methyl-6- (pyridin-3-yl)-5-(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)amino)benzamide) 442 34-3

N-hydroxy-3- (methyl(1-methyl-6- phenyl-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 441

Example 35-1.N-hydroxy-3-((1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

Step 1. Methyl3-((5-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate

A 25 mL reaction vial was charged with4-bromo-5-(trifluoromethyl)benzene-1,2-diamine (700 mg, 2.74 mmol), DMF(8 mL), triethylamine (1.4 mL, 10 mmol) and methyl3-isothiocyanatobenzoate (530 mg, 2.74 mmol), and the mixture wasstirred at room temperature for 15 minutes.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.06 g, 6.85 mmol) wasadded, and the resulting reaction mixture was stirred at roomtemperature for 30 min then at 90° C. for 4 h. DMF was removed underreduce pressure, and ethyl acetate (10 mL) and brine (5 mL) were addedto the residue. The mixture was shaken and the layers were separated.The aqueous phase was extracted with ethyl acetate (10 mL), and thecombined organic layers were evaporated under reduce pressure. Theresidue purified via column chromatography on silica gel (eluting with2:1 ethyl acetate/hexanes) to give methyl3-((5-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(706 mg, 62%) as white solid. MS: (ESI, m/z): 415 [M+H]⁺.

Step 2. (methyl3-((5-bromo-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoateand methyl3-((6-bromo-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate)

A 4 mL reaction vial was charged with methyl3-((5-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(100 mg, 0.242 mmol), acetonitrile (2 mL) and iodomethane (1M inacetonitrile, 315 μL, 0.315 mmol). Cesium carbonate (95 mg, 0.29 mmol)was added, and the mixture was shaken at room temperature overnight. Thesolvent was evaporated under reduce pressure, the residue was dilutedwith ethyl acetate (2 mL) and brine (1 mL), and the mixture was shaken.The organic layer was separated and the aqueous phase was extracted withethyl acetate (1 mL). The combined organic layers were concentratedunder a stream of nitrogen. The crude product was purified via columnchromatography on silica gel (eluting with 2:1 ethyl acetate/hexanes) toafford a mixture of methyl3-((5-bromo-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoateand methyl3-((6-bromo-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate)(92 mg, 89%) as a white solid. MS: (ESI, m/z): 429[M+H]⁺.

Step 3. methyl3-((1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoateand methyl3-((1-methyl-6-(pyridin-3-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate

A 4 mL reaction vial was charged with the mixture of methyl3-((5-bromo-1-methyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoateand methyl3-((6-bromo-1-methyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(42.8 mg, 100 μmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (51.2 mg, 250μmol), 1,4-dioxane (600 μL) and water (60 μL). Potassium phosphatetribasic (2 M in water, 200 μL, 400 μmol) and Pd(PPh₃)₄(11.6 mg, 10μmol) were added, and the vial was sealed and shaken at 120° C.overnight. Ethyl acetate (1 mL) and brine (500 μL) were added, and themixture was shaken. The organic layer was separated and the aqueousphase was extracted with ethyl acetate (1 mL). The combined organiclayers were concentrated under a stream of nitrogen and the crudeproduct was purified by preparative thin layer chromatography (elutingwith 4:1 ethyl acetate/hexanes). The top band on TLC plate was assumedas methyl3-((1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(6.2 mg, 14%) MS: (ESI, m/z): 427[M+H]⁺. The lower band was assumed asmethyl3-((1-methyl-6-(pyridin-3-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(8.1 mg, 18.7%), MS: (ESI, m/z): 427[M+H]⁺.

Step 4.N-hydroxy-3-((1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

A 2 mL reaction vial was charged with methyl3-((1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(6.2 mg, 14 μmol) and THF/methanol (3:1, 200 μL), and the vial wassealed and shaken at 50° C. for 15 minutes and then cooled to roomtemperature. Hydroxylamine (50% v/v solution in water, 150 μL,) wasadded, followed by 1 N aqueous NaOH solution (100 μL). The vial wassealed and then shaken at room temperature for 16 h. The reactionmixture was concentrated under a stream of nitrogen at room temperature,dissolved in 500 μL of DMSO, and purified by prep-HPLC with thefollowing conditions: Column: Waters SunFire C18, 5 um, 19×150 mm;mobile phase, A: water with 0.1% formic acid, B: CH₃CN with 0.1% formicacid; Flow rate, 23 mL/min; Gradient, 0% B to 5% B in 5 min; Detector,254, 220 nm. The collected fractions were concentrated to giveN-hydroxy-3-((1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(1.3 mg, 20.9%) MS: (ESI, m/z): 428[M+H]⁺.

The following compounds were prepared according to the proceduresoutlinedN-hydroxy-3-((1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide:

(ESI, m/z) Example Structure Name [M + H]+ 35-2

N-hydroxy-3-((1- methyl-6-(pyridin-3- yl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 35-3

N-hydroxy-3-((1- methyl-6-phenyl-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 427

Example 36-1.N-hydroxy-3-((5-phenyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

A 2 mL reaction vial was charged with methyl3-((5-bromo-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzoate(0.2 M in 1,4-dioxane, 150 μL, 30 μmol), phenylboronic acid (0.2 M in1,4-dioxane, 275 μL, 45 μmol), potassium phosphate tribasic (1 M inwater, 120 μL, 120 μmol), and Pd(PPh₃)₄(0.02 M in toluene, 75 μL, 1.5μmol). The vial was sealed and heated at 110° C. overnight. The mixturewas concentrated under a stream of N₂. The residue was diluted withethyl acetate (0.7 mL) and brine (0.5 mL), and the mixture was shaken.The organic layer was separated and the aqueous phase was extracted withethyl acetate (0.7 mL). The combined organic layers were concentratedunder a stream of nitrogen, and THF/methanol (3:1, 200 μL) was added tothe residue. The vial was sealed and shaken at 50° C. for 15 minutes todissolve the residue. Hydroxylamine (50% v/v solution in water, 150 μL)was added followed by 1 N aqueous NaOH solution (100 μL). The vial wassealed and shaken at room temperature for 16 h, and then concentratedunder a stream of nitrogen at room temperature. The residue wasdissolved in 500 μL of DMSO and purified by prep-HPLC with the followingconditions: Column: Waters SunFire C18, 5 um, 19×150 mm; mobile phase,A: water with 0.1% formic acid, B: CH₃CN with 0.1% formic acid; Flowrate, 23 mL/min; Gradient, 0% B to 5% B in 5 min; Detector, 254, 220 nm.The collected fractions were concentrated to giveN-hydroxy-3-((5-phenyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(2.6 mg, 20.6% yield) as an off-white solid. MS: (ESI, m/z): 413[M+H]⁺.

The following compounds were prepared according to the proceduresoutlinedN-hydroxy-3-((1-methyl-5-(pyridin-3-yl)-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide:

(ESI, m/z) Example Structure Name [M + H]+ 36-2

N-hydroxy-3-((6- (pyridin-3-yl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 414 36-3

N-hydroxy-3-((5- (pyridin-4-yl)-6- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 414 36-4

3-((6-(3- ethoxyphenyl)-5- (trifluoromethyl)-1H- benzo[d]imdiazol-2-yl)amino)-N- hydroxybenzamide 457 36-5

N-hydroxy-3-((6-(4- (methylthio)phenyl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 459 36-6

4-(2-((3- (hydroxycarbamoyl) phenyl)amino)-5- (trifluoromethyl)-1H-benzo[d]imidazol-6- yl)-N,N- dimethylbenzamide 484 36-7

N-hydroxy-3-((6-(4- (methoxymethyl) phenyl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 457 36-8

N-hydroxy-3-((6- (quinolin-6-yl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 464 36-9

3-((6-(2,3- dihydrobenzo[d][1,4] dioxin-6-yl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)-N- hydroxybenzamide 471 36-10

N-hydroxy-3-((6-(3- hydroxyphenyl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 429 36-11

N-hydroxy-3-((6-(2- (hydroxymethyl)phenyl)- 5-(trifluoromethyl)-1H-benzo[d]imidazol- 2-yl)amino)benzamide 443 36-12

3-((6-(6- (dimethylamino)pyridin- 3-yl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)-N- hydroxybenzamide 457 36-13

N-hydroxy-3-((6-(2- morpholinopyrimidin- 5-yl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 500 36-14

N-hydroxy-3-((6-(3- (morpholine-4- carbonyl)phenyl)-5-(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)amino)benzamide 526 36-15

3-((6-(5-fluoro-2- (hydroxymethyl)phenyl)- 5-(trifluoromethyl)-1H-benzo[d]imidazol- 2-yl)amino)-N- hydroxybenzamide 461 36-16

N-hydroxy-3-((6-(3- (methoxymethyl) phenyl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 457 36-17

N-hydroxy-3-((6-(1- methyl-1H-indazol-6- yl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 467 36-18

3-(2-((3- (hydroxycarbamoyl) phenyl)amino)-5- (trifluoromethyl)-1H-benzo[d]imidazol-6- yl)-N,N- dimethylbenzamide 484 36-19

N-hydroxy-3-((6-(4- mnorpholinophenyl)-5- (trifluoromethyl)-1H-benzo[d]imidazol-2- yl)amino)benzamide 498 36-20

3-((6-(furan-3-yl)-5- (trifluoromethyl)-1H- benzo[d]imidazol-2-yl)amino)-N- hydroxybenzamide 403

Example 37-1.N-((benzylcarbamoyl)oxy)-3-((5-phenyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide

A 2 mL reaction vial was charged withN-hydroxy-3-((5-phenyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(12.4 mg, 30 μmol), acetonitrile (600 μL), and 1,1′-carbonyldiimidazole(6.3 mg, 39 μmol). The mixture was shaken at room temperature for 30minutes and then benzylamine (4.8 mg, 45 μmol) was added. The vial wasshaken at room temperature for 16 h and then concentrated under a streamof nitrogen. The residue was dissolved in 500 μL of DMSO and purified byprep-HPLC with the following conditions: Column: Waters SunFire C18, 5um, 19×150 mm; mobile phase, A: water with 0.1% formic acid, B: CH₃CNwith 0.1% formic acid; Flow rate, 23 mL/min; Gradient, 0% B to 5% B in 5min; Detector, 254, 220 nm. The collected fractions were concentrated toyieldN-((benzylcarbamoyl)oxy)-3-((5-phenyl-6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)amino)benzamide(4 mg, 24%) as an off-white solid. MS: (ESI, m/z): 546[M+H]⁺.

The following compound was synthesized according to the above protocol:

(ESI, m/z) Example Structure Name [M + H]+ 37-2

N-((benzylcarbamoyl)oxy)-3- ((5-cyano-6-(trifluoromethyl)-1H-benzo[d]imidazol-2- yl)(methyl)amino)benzamide 509

Example 38-1. In Vitro Histone Deacetylase Assay

The probe binding HDAC11 assay was performed using a time resolvedfluorescence (TRF) assay format. Recombinant N-terminal GST tagfull-length human HDAC11 was expressed and purified from baculovirus inSf9 insect cells (SignalChem, # H93-30G-1000). Each assay was performedin 1536 black well microplates (Corning, #3936) in a final volume of 8μL in assay buffer containing 50 mM HEPES (pH 7.5), 50 mM KCl, 50 mMNaCl, 0.5 mM GSH (L-Glutathione reduced, Sigma #G4251), 0.03% BGG (0.22μM filtered, Sigma, #G7516-25G), and 0.01% Triton X-100 (Sigma,#T9284-10L). 100 nL of 10-point, 3-fold serial dilution in DMSO waspre-dispensed into respective wells of 1536 assay plates for a finaltest concentration range of 25 μM to 1.3 nM respectively. The finalconcentration in the assay of HDAC11 and probe (a fluorescein labeledHDAC11 inhibitor) was 2.5 nM and 20 nM respectively. 4 μL of 2× probeand 2× anti-GST Terbium (Cisbio, #61GSTXLB) was added to assay platesfollowed by 4 μL of 2×HDAC11. Plates were incubated for 16 hours at roomtemperature before time resolved fluorescence was read on the Envision(Excitation at 340 nm, and Emission at 485 nm and 535 nm, Perkin Elmer).

Data from HDAC11 Assays were reported as percent inhibition (inh)compared with control wells based on the following equation: %inh=1−((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured time resolvedfluorescence. AveLow=average time resolved fluorescence of no enzymecontrol (n=32). AveHigh=average time resolved fluorescence of DMSOcontrol (n=32). IC50 values were determined by curve fitting of thestandard 4 parameter logistic fitting algorithm included in the ActivityBase software package: IDBS XE Designer Model205. Data is fitted usingthe Levenburg Marquardt algorithm.

As set forth in Table I-2 below, “+++” indicates an IC₅₀ below 0.5 M;“++” indicates an IC₅₀ between 0.5 μM and 1 μM; and “+” indicates anIC₅₀ above 1 μM.

TABLE I-2 IC₅₀ Ranges for Compounds of the Disclosure Compound HDAC11No. IC₅₀ Range  1-1 +++  2-1 +++  3-1 +++  4-1 +++  4-2 +++  4-3 +++ 4-5 +++  4-6 +++  4-7 +++  4-8 +  5-1 +++  6-1 ++  7-1 +++  8-1 +  9-1+++ 10-1 +++ 11-1 +++ 12-1 +++ 13-1 +++ 14-1 +++ 15-1 +++ 16-1 +++ 16-2+++ 17-1 +++ 18-1 +++ 19-1 +++ 20-1 +++ 20-2 +++ 21-1 +++ 22-1 +++ 22-2+++ 23-1 + 23-2 + 23-3 + 23-4 +++ 24-1 + 24-2 + 25-1 + 26-1 + 27-1 +++27-2 +++ 28-1 +++ 29-1 + 30-1 + 31-1 +++ 32-1 + 32-2 +++ 33-1 +++ 33-2+++ 33-3 ++ 33-4 + 33-5 +++ 33-6 +++ 33-7 +++ 33-8 + 33-9 ++ 33-10 +++33-11 +++ 33-12 +++ 33-13 + 33-14 +++ 33-15 +++ 33-16 +++ 33-17 +++33-18 + 33-19 ++ 33-20 + 33-21 ++ 33-22 +++ 33-23 + 33-24 + 34-1 +34-2 + 34-3 ++ 35-1 ++ 35-2 +++ 35-3 +++ 36-1 +++ 36-2 +++ 36-3 +++ 36-4+++ 36-5 +++ 36-6 +++ 36-7 +++ 36-8 +++ 36-9 +++ 36-10 +++ 36-11 +++36-12 +++ 36-13 +++ 36-14 +++ 36-15 +++ 36-16 +++ 36-17 +++ 36-18 +++36-19 +++ 36-20 +++ 37-1 +++ 37-2 +

Synthesis of Spiro Compounds Example 1-2—Preparation of1′-(4-chlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-1)

Step-1: Methyl 1-oxo-2,3-dihydro-1H-indene-4-carboxylate

Into a 1000-mL pressure tank reactor was placed4-bromo-2,3-dihydro-1H-inden-1-one (30 g, 142 mmol, 1 equiv), MeOH (450mL), Pd(dppf)Cl₂.CH₂Cl₂ (25 g, 0.1 equiv), and Et₃N (150 mL). Theresulting mixture was stirred for 24 h at 100° C. under an atmosphere of50 MPa CO (g). After cooling to room temperature, the mixture wasfiltered through a pad of celite and then concentrated under vacuum. Theresidue was purified by normal phase chromatography on silica gel usingEtOAc/petroleum ether (1:10). The collected fractions were concentratedunder vacuum to afford 17 g (63% yield) of the title compound as a whitesolid. MS: (ES, m/z): 191 [M+H]⁺.

Step-2: Dimethyl 1-oxo-2,3-dihydro-1H-indene-2,4-dicarboxylate

Into a 500-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed dimethyl carbonate (180 mL) andmethyl 1-oxo-2,3-dihydro-1H-indene-4-carboxylate (30 g, 0.158 mol, 1equiv). To this mixture, NaH (60% dispersion in oil, 12.6 g, 0.316 mol,2 equiv) was added at room temperature. The resulting solution wasstirred for 2 h at 80° C. The reaction was quenched by the addition of40 mL of water and extracted with 3×200 mL of EtOAc. The combinedorganic layers were dried over Na₂SO₄ and concentrated under vacuum. Theresidue was purified by normal phase chromatography on silica gel usingEtOAc/petroleum ether (1:10). The collected fractions were concentratedunder vacuum to afford 8 g of the title compound as a white solid. MS:(ES, m/z): 249 [M+H]⁺.

Step-3: Dimethyl2-(cyanomethyl)-1-oxo-2,3-dihydro-1H-indene-2,4-dicarboxylate

Into a 500-mL 3-necked round-bottom flask was placed dimethyl1-oxo-2,3-dihydro-1H-indene-2,4-dicarboxylate (7 g, 28.2 mmol, 1 equiv),Et₃N (15 g, 148 mmol, 5 equiv), 2-bromoacetonitrile (10 g, 83 mmol, 3equiv) and THF (200 mL). The resulting solution was stirred for 16 h atroom temperature and then concentrated under vacuum. The residue wasdiluted with 250 mL of water and extracted with 3×100 mL of EtOAc. Thecombined organic layers were dried over Na₂SO₄ and concentrated undervacuum. The residue was purified by normal phase chromatography onsilica gel using EtOAc/petroleum ether (1:10). The collected fractionswere concentrated under vacuum to afford 5 g (62% yield) of the titlecompound as a white solid. MS: (ES, m/z): 288 [M+H]⁺.

Step-4: Dimethyl2-(2-aminoethyl)-1-hydroxy-2,3-dihydro-1H-indene-2,4-dicarboxylateacetate

Into a 250-mL round-bottom flask equipped with a balloon of H₂ (g), wasplaced a solution of dimethyl2-(cyanomethyl)-1-oxo-2,3-dihydro-1H-indene-2,4-dicarboxylate (3 g,10.44 mmol, 1 equiv) in MeOH (100 mL), AcOH (60 mL), and PtO₂ (1.28 g).The resulting solution was stirred for 16 h at room temperature under anatmosphere of H₂ (g). The resulting mixture was filtered. The filtratewas concentrated under vacuum to give 3.7 g (crude) of the titlecompound as yellow oil which was used without further purification. MS:(ES, m/z): 294 [M+H]⁺.

Step-5: Methyl1-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 250-mL round-bottom flask was placed a solution of dimethyl2-(2-aminoethyl)-1-hydroxy-2,3-dihydro-1H-indene-2,4-dicarboxylateacetate (3.7 g, 10.48 mmol, 1 equiv) in MeOH (60 mL), and NH₃ solutionin MeOH (40 mL). The resulting solution was stirred for 4 h at roomtemperature and then concentrated under vacuum. The residue was purifiedby normal phase chromatography on silica gel using MeOH/CH₂Cl₂ (1:10).The collected fractions were concentrated under vacuum to afford 1.3 g(48% yield) of the title compound as a white solid. MS: (ES, m/z): 262[M+H]⁺.

Step-6: Methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 250-mL round-bottom flask was placed methyl1-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(1.3 g, 4.98 mmol, 1 equiv), triethylsilane (10 mL) and TFA (60 mL). Theresulting solution was stirred for 48 h at room temperature and thenconcentrated under vacuum. The residue was purified by normal phasechromatography on silica gel using MeOH/CH₂Cl₂ (1:10). The collectedfractions were concentrated under vacuum to afford 700 mg (57% yield) ofthe title compound as a white solid. ¹H NMR (300 MHz, DMSO-d6) δ (ppm):7.76-7.73 (d, J=7.2 Hz, 2H), 7.47-7.45 (d, J=7.2 Hz, 1H), 7.32-7.27 (t,J=7.6 Hz, 1H), 3.82 (s, 3H), 3.39-3.12 (m, 5H), 2.92-2.87 (d, J=16.2 Hz,1H), 2.03-1.90 (m, 2H). MS: (ES, m/z): 246 [M+H]⁺.

Step-7:1′-(4-Chlorobenzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylicacid

Into a 50-mL round-bottom flask was placed a solution of methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (60 mg,0.24 mmol, 1 equiv) in DMF (3 mL), and NaH (60% dispersion in oil, 30mg, 0.75 mmol, 3.12 equiv) was added at room temperature. The mixturewas stirred for 20 min at room temperature and1-(bromomethyl)-4-chlorobenzene (48 mg, 0.23 mmol, 0.95 equiv) wasadded. The resulting solution was stirred for 3 h at room temperature.The reaction was then quenched by the addition of 0.5 mL of water. Thecrude product was purified by reverse phase chromatography with thefollowing conditions: Column: C18, 40 g, 20-35 μm; Mobile Phase A:Water/0.05% formic acid, Mobile Phase B: CH₃CN; Flow rate: 40 mL/min;Gradient: 5% B to 70% B in 25 min; Detector: UV 254 nm. The collectedfractions were lyophilized to afford 50 mg (57% yield) of the titlecompound as a white solid. MS: (ES, m/z): 356 [M+H]⁺.

Step-8:1′-(4-Chlorobenzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed a solution of1′-[(4-chlorophenyl)methyl]-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylicacid (50 mg, 0.14 mmol, 1 equiv) in DMF (3 mL), isopropyl chloroformate(17 mg, 0.14 mmol, 1 equiv), and NMM (14 mg, 0.14 mmol, 0.98 equiv). Themixture was stirred for 1 h and NH₂OH.HCl (30 mg, 0.43 mmol, 3.1 equiv)was added. The resulting solution was stirred overnight at roomtemperature. The crude product was purified by Prep-HPLC with thefollowing conditions: Column: X Bridge C18, 19×150 mm, 5 μm; MobilePhase A: Water/0.05% TFA, Mobile Phase B: CH₃CN; Flow rate: 20 mL/min;Gradient: 30% B to 70% B in 10 min; Detector: UV 254 nm. The collectedfractions were lyophilized to afford 16 mg (28% yield) of the titlecompound as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 10.91 (s,1H), 7.46-7.42 (m, 2H), 7.34-7.31 (m, 2H), 7.28-7.20 (m, 3H), 4.47-4.39(m, 2H), 3.36-2.87 (m, 6H), 2.07-1.94 (m, 2H). MS: (ES, m z): 371[M+H]⁺.

TABLE 1-2 Found (ES, m/z) Ex. Structure 1H-NMR δ (ppm) [M + H]⁺ II-2

(400 MHz, DMSO-d6): 10.90 (br s, 1H), 7.33-7.31 (m, 2H), 7.24-7.20 (m,1H), 3.35-3.26 (m, 3H), 3.14 (d, J = 16 Hz, 1H), 3.02 (d, J = 16 Hz,1H), 2.85 (d, J = 16 Hz, 1H), 2.80 (s, 3H), 1.94 (t, J = 5.8 Hz, 2H) 261The following compound was prepared according to the method of Example1-2, with the following modification: In Step 7, the halide used wasmethyl iodide.

Example 2-2—Preparation of(R)-1′-(4-chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamideand(S)-1′-(4-chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-3 and HDTK054)

Step-1: Methyl1′-(3-chloro-4-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed a solution of methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (300 mg,1.22 mmol, 1 equiv) in THF (10 mL). Then NaH (60% dispersion in oil, 100mg, 2.50 mmol, 2 equiv) was added at 0° C. over 15 min, followed by4-(bromomethyl)-1-chloro-2-(trifluoromethyl)benzene (655 mg, 2.40 mmol,2 equiv). The resulting solution was stirred for 3 h at room temperatureand then concentrated under vacuum. The residue was purified by normalphase chromatography on silica gel using EtOAc/petroleum ether (2:1).The collected fractions were concentrated under vacuum to afford 300 mg(56% yield) of the title compound as a white solid. MS: (ES, m/z): 438[M+H]⁺.

Step-2: Chiral separation of methyl(R)-1′-(4-chloro-3-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylateand methyl(S)-1′-(4-chloro-3-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

The racemate of methyl1′-(3-chloro-4-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylatewas separated by Prep-SFC with the following conditions: Column:Chiralpak IA 2×25 cm, 5 μm; Mobile Phase A:CO₂, 70%, Mobile Phase B:MeOH, 30%; Flow rate: 40 mL/min; Detector: UV 220 nm. The first elutingisomer (Rt 4.87 min) was collected and concentrated under vacuum to give110 mg (56% yield) of a white solid which was assigned as the R isomerof methyl1′-(3-chloro-4-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate.MS: (ES, m/z): 438 [M+H]⁺. The second eluting isomer (Rt 6.24 min) wascollected and concentrated under vacuum to give 120 mg (61% yield) of awhite solid which was assigned as the S isomer of methyl1′-(3-chloro-4-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate.MS: (ES, m/z): 438 [M+H]⁺.

Step-3:(R)-1′-(4-Chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed a solution of the firsteluted isomer from Step 2, which was assigned as methyl(R)-1′-(4-chloro-3-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylateas described above, (110 mg, 0.25 mmol, 1 equiv) in THF/MeOH (4:1, 2mL), NH₂OH (50% in H₂O, 2 mL, 30.21 mmol, 120 equiv), and aq. 1N NaOH(0.50 mL, 0.50 mmol, 2 equiv). The resulting solution was stirred for 16h at room temperature. The solids were filtered out. The crude productwas purified by Prep-HPLC with the following conditions: Column: XBridgePrep C18 OBD, 19×150 mm, 5 μm; Mobile Phase A: water/0.1% formic acid,Mobile Phase B: CH₃CN; Flow rate: 20 mL/min; Gradient: 5% B to 80% B in8 min; Detector: UV 254 nm, 220 nm. The collected fractions werelyophilized to give 48 mg (44% yield) of the title compound as anoff-white solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 10.91 (br s, 1H),8.97 (br s, 1H), 7.74 (d, J=8.8 Hz, 2H), 7.55 (d, J=8.4 Hz, 1H),7.33-7.32 (m, 2H), 7.24-7.21 (m, 1H), 4.58-4.48 (m, 2H), 3.37-3.25 (m,3H), 3.18-3.08 (m, 2H), 2.92 (d, J=16.0 Hz, 1H), 1.98 (t, J=6.4 Hz, 2H).MS: (ES, m/z): 439 [M+H]⁺. Chiral HPLC (Column: Chiralpak AS-3, 0.46×5cm, 3 μm; Mobile Phase A: hexanes/0.5% TFA, Mobile Phase B: EtOH; Flowrate: 1 mL/min; Gradient: 20% B hold for 8 min; Detector: UV 254 nm): Rt2.25 min; >99%.

Step-4:(S)-1′-(4-Chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

The procedure from Step 3 was followed using the second eluted isomerfrom Step 2, which was assigned as methyl(S)-1′-(4-chloro-3-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylateas described above, (120 mg, 0.27 mmol, 1 equiv) to afford 51.3 mg (43%yield) of the title compound as an off-white solid. ¹H NMR (400 MHz,DMSO-d6) δ (ppm): 10.91 (br s, 1H), 8.97 (br s, 1H), 7.75 (d, J=8.8 Hz,2H), 7.55 (d, J=8.4 Hz, 1H), 7.33-7.32 (m, 2H), 7.24-7.21 (m, 1H),4.58-4.48 (m, 2H), 3.37-3.25 (m, 3H), 3.20-3.06 (m, 2H), 2.92 (d, J=16.0Hz, 1H), 1.98 (t, J=6.4 Hz, 2H). MS: (ES, m/z): 439 [M+H]⁺. Chiral HPLC:Rt 3.94 min; >99%.

Example 3-2—Preparation of1′-(3-fluoro-4-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-5)

Step-1: Methyl1′-(3-fluoro-4-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed a solution of methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (50 mg,0.20 mmol, 1 equiv) in THF (5 mL). Then NaH (60% dispersion in oil, 10mg, 0.25 mmol, 1.25 equiv) was added at room temperature. After stirringfor 15 min, 4-(bromomethyl)-2-fluoro-1-(trifluoromethyl)benzene (60 mg,0.23 mmol, 1.2 equiv) was added. The resulting solution was stirred for2 h at room temperature and then concentrated under vacuum. The residuewas purified by normal phase chromatography on silica gel usingEtOAc/petroleum ether (1:1). The collected fractions were concentratedunder vacuum to afford 48 mg (56% yield) of the title compound as acolorless oil. MS: (ES, m/z): 422 [M+H]⁺.

Step-2:1′-(3-Fluoro-4-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed methyl1′-(3-fluoro-4-(trifluoromethyl)benzyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(43 mg, 0.10 mmol, 1 equiv), THF/MeOH (1:1, 3 mL), NH₂OH (50% in H₂O,0.2 mL), and aq. NaOH (8 mg in 0.2 mL). The resulting solution wasstirred for 2 h at room temperature. The crude product was purified byPrep-HPLC with the following conditions: Column: XBridge Prep C18,19×150 mm, 5 μm; Mobile Phase A: water/0.05% TFA, Mobile Phase B: CH₃CN;Flow rate: 20 mL/min; Gradient: 10% B to 80% B in 15 min; Detector: UV254 nm. The collected fractions were lyophilized to afford 10.2 mg (24%yield) of the title compound as a white solid. ¹H NMR (400 MHz, DMSO-d6)δ (ppm): 10.91 (s, 1H), 9.00 (br s, 1H), 7.91 (t, J=7.6 Hz, 2H),7.42-7.20 (m, 5H), 4.55 (s, 2H), 3.47-3.28 (m, 4H), 3.20 (d, J=16 Hz,1H), 3.10 (d, J=16 Hz, 1H), 2.94 (d, J=16 Hz, 1H), 2.01 (t, J=8.4 Hz,2H). MS: (ES, m/z): 423 [M+H]⁺.

TABLE 2-2 Found (ES, m/z) Ex. Structure ¹H-NMR δ (ppm) [M + H]⁺ II-6

(400 MHz, DMSO-d6): 10.90 (br s, 1H), 9.08 (br s, 1H), 7.76-7.74 (m,2H), 7.48-7.45 (m, 2H), 7.35-7.31 (m, 2H), 7.24-7.20 (m, 1H), 4.55 (t, J= 3.6 Hz, 2H), 3.35-3.26 (m, 3H), 3.20 (d, J = 16 Hz, 1H), 3.08 (d, J =16.8 Hz, 1H), 2.92 (d, J = 16 Hz, 1H), 1.98 (t, J = 6.4 Hz, 2H) 405 II-7

(400 MHz, DMSO-d6): 10.92 (br s, 1H), 9.03-8.90 (m, 1H), 7.34-7.13 (m,7H), 4.48 (s, 2H), 3.34 (d, J = 16.8 Hz, 1H), 3.24-3.16 (m, 3H), 3.06(d, J = 17.2 Hz, 1H), 2.89 (d, J = 16 Hz, 1H), 1.95-1.93 (m, 2H) 355II-8

(400 MHz, DMSO-d6): 9.00 (br s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.51 (d,J = 8.4 Hz, 2H), 7.35-7.32 (m, 2H), 7.25- 7.21 (m, 1H), 4.56 (s, 2H),3.39-3.07 (m, 8H), 2.94 (d, J = 17 Hz, 1H), 2.00 (t, J = 6.8 Hz, 2H) 415The following compounds were prepared according to the method of Example3-2, with the following modification: In Step 2, the solvent system canbe MeOH or a 1:1 mixture of THF/MeOH.

Example 4-2—Preparation of1′-cyclopropyl-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-9)

Step-1: Methyl1′-cyclopropyl-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 50-mL round-bottom flask was placed methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (50 mg,0.20 mmol, 1 equiv), cyclopropylboronic acid (150 mg, 1.75 mmol, 10equiv), Cu(OAc)₂ (100 mg, 0.55 mmol, 2.7 equiv), Et₃N (150 mg, 1.48mmol, 7.27 equiv), pyridine (75 mg, 0.95 mmol, 4.65 equiv), and THF (10mL). The resulting solution was stirred for 48 h at 60° C. The solidswere filtered out and the filtrate was concentrated under vacuum. Theresidue was purified by normal phase chromatography on silica gel withEtOAc/petroleum ether (1:1). The collected fractions were concentratedto give 38 mg (65% yield) of the title compound as a white solid. MS:(ES, m/z): 286 [M+H]⁺.

Step-2:1′-Cyclopropyl-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed a solution of methyl1′-cyclopropyl-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(38 mg, 0.13 mmol, 1 equiv) in THF/MeOH (4:1, 3 mL), NH₂OH (50% in H₂O,0.2 mL, 3.03 mmol, 23 equiv), and aq. 1N NaOH (0.27 mL, 0.27 mmol, 2equiv). The resulting solution was stirred for 2 h at room temperature.The solids were filtered out. The crude product was purified byPrep-HPLC with the following conditions: Column: SunFire Prep C18 OBD,19×150 mm, 5 μm; Mobile Phase A: water/0.1% formic acid, Mobile Phase B:CH₃CN; Flow rate: 20 mL/min; Gradient: 5% B to 48% B in 10 min;Detector: UV 254 nm, 220 nm. The collected fractions were lyophilized togive 2.8 mg (7% yield) of the title compound as a white solid. ¹H NMR(400 MHz, DMSO-d6) δ (ppm): 10.90 (s, 1H), 8.98 (s, 1H), 7.32 (d, J=7.2Hz, 2H), 7.22 (t, J=7.2 Hz, 1H), 3.29-3.22 (m, 3H), 3.14 (d, J=16 Hz,1H), 3.02 (d, J=16 Hz, 1H), 2.84 (d, J=16 Hz, 1H), 2.74-2.70 (m, 1H),1.90 (t, J=6.8 Hz, 2H), 0.69-0.67 (m, 4H). MS: (ES, m/z): 287 [M+H]⁺.

Example 5-2—Preparation of1′-(cyclobutylmethyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-10)

Step-1: Cyclobutylmethyl1′-(cyclobutylmethyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 10-mL vial was placed methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (80 mg,0.33 mmol, 1 equiv), (bromomethyl)cyclobutane (480 mg, 3.22 mmol, 10equiv), NaO^(t)Bu (80 mg, 0.71 mmol, 2.16 equiv), and DMF (3 mL). Thereaction mixture was heated at 100° C. for 1 h in a microwave reactor.After cooling, the solids were filtered out. The crude product waspurified by reverse phase chromatography with the following conditions:Column: C18, 40 g, 20-35 μm; Mobile Phase A: Water/0.05% TFA, MobilePhase B: CH₃CN; Flow rate: 40 mL/min; Gradient: 5% B to 95% B in 35 min;Detector: UV 254 nm. The collected fractions were lyophilized to give 40mg (33% yield) of the title compound as a white solid. MS: (ES, m/z):368 [M+H]⁺.

Step-2:1′-(Cyclobutylmethyl)-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed a solution ofcyclobutylmethyl1′-(cyclobutylmethyl)-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(40 mg, 0.11 mmol, 1 equiv) in THF/MeOH (4:1, 3 mL), NH₂OH (50% in H₂O,0.72 mL, 10.9 mmol, 100 equiv), and aq. 1N NaOH (0.44 mL, 0.44 mmol, 4equiv). The resulting solution was stirred for 10 h at room temperature.The solids were filtered out. The crude product was purified byPrep-HPLC with the following conditions: Column: SunFire Prep C18 OBD,19×150 mm, 5 μm; Mobile Phase A: water/0.1% formic acid, Mobile Phase B:CH₃CN; Flow rate: 20 mL/min; Gradient: 5% B to 85% B in 10 min;Detector: UV 254 nm, 220 nm. The collected fractions were lyophilized togive 3.0 mg (8% yield) of the title compound as a white solid. ¹H NMR(400 MHz, DMSO-d6) δ (ppm): 10.90 (s, 1H), 8.97 (s, 1H), 7.33-7.30 (m,2H), 7.24-7.21 (m, 1H), 3.31-3.25 (m, 4H), 3.14 (d, J=16 Hz, 1H), 3.02(d, J=16 Hz, 1H), 2.85 (d, J=16 Hz, 1H), 2.52-2.50 (m, 1H), 2.03-1.81(m, 7H), 1.73-1.61 (m, 2H). MS: (ES, m/z): 315 [M+H]⁺.

Example 6-2—Preparation ofN-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-11)

Step-1: Methyl2′-oxo-1′-(4-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 50-mL round-bottom flask was placed methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (100 mg,0.41 mmol, 1 equiv), 1-iodo-4-(trifluoromethyl)benzene (100 mg, 0.37mmol, 1.2 equiv), methyl[2-(methylamino)ethyl]amine (40 mg, 0.45 mmol,0.4 equiv), CuI (40 mg, 0.21 mmol, 0.2 equiv), Cs₂CO₃ (400 mg, 1.23mmol, 3 equiv), and DMSO (15 mL). The resulting solution was stirred for12 h at 130° C. After cooling, 10 mL of H₂O was added to the solution.The resulting solution was extracted with 20 mL of EtOAc, dried overNa₂SO₄, and concentrated under vacuum. The residue was purified bynormal phase chromatography on silica gel with EtOAc/petroleum ether(1:10). The collected fractions were concentrated to give 70 mg (44%yield) of the title compound as a yellow solid. MS: (ES, m/z): 390[M+H]⁺.

Step-2:2′-Oxo-1′-(4-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylicacid

Into a 25-mL round-bottom flask was placed a solution of methyl2′-oxo-1′-(4-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(50 mg, 0.13 mmol, 1 equiv) in THF (10 mL), and NaOH (40 mg, 1.00 mmol,7.6 equiv). The resulting solution was stirred for 18 h at 70° C. The pHvalue of the solution was adjusted to 4 with 1N HCl. The solids werefiltered out. The crude product was purified by reverse phasechromatography with the following conditions: Column: C18, 40 g, 20-35μm; Mobile Phase A: Water/0.05% TFA, Mobile Phase B: CH₃CN; Flow rate:40 mL/min; Gradient: 25% B to 50% B in 10 min; Detector: UV 254 nm. Thecollected fractions were lyophilized to give 20 mg (41% yield) of thetitle compound as a white solid. MS: (ES, m/z): 376 [M+H]⁺.

Step-3:N-Hydroxy-2′-oxo-1′-(4-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed a solution of2′-oxo-1′-(4-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylicacid (50 mg, 0.13 mmol, 1 equiv) in DMA (3 mL), NMM (10 mg, 0.10 mmol, 1equiv), isopropyl chloroformate (10 mg, 0.08 mmol, 0.8 equiv) andNH₂OH.HCl (100 mg, 1.45 mmol, 14.5 equiv). The resulting solution wasstirred for 12 h at 25° C. The solids were filtered out. The crudeproduct was purified by Prep-HPLC with the following conditions: Column:XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile Phase A: water/0.1% formicacid, Mobile Phase B: CH₃CN; Flow rate: 20 mL/min; Gradient: 5% B to 60%B in 8 min; Detector: UV 254 nm, 220 nm. The collected fractions werelyophilized to give 16 mg (32% yield) of the title compound as a lightyellow solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 10.91 (br s, 1H), 9.01(br s, 1H), 7.97 (d, J=8.4 Hz, 2H), 7.77 (d, J=8.4 Hz, 2H), 7.37-7.34(m, 2H), 7.28-7.23 (m, 1H), 3.93 (t, J=6.4 Hz, 2H), 3.44 (d, J=16.1 Hz,1H), 3.33-3.19 (m, 2H), 3.05 (d, J=16.1 Hz, 1H), 2.15 (t, J=6.4 Hz, 2H).MS: (ES, m/z): 391 [M+H]⁺.

Example 7-2—Preparation ofN-hydroxy-2′-oxo-1′-(3-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-12)

Step-1: Methyl2′-oxo-1′-(3-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (200 mg,0.82 mmol, 1 equiv), 1-iodo-3-(trifluoromethyl)benzene (200 mg, 0.74mmol, 1.2 equiv), CuI (40 mg, 0.21 mmol, 0.2 equiv),methyl[2-(methylamino)ethyl]amine (40 mg, 0.45 mmol, 0.4 equiv), Cs₂CO₃(400 mg, 1.23 mmol, 3 equiv), and DMSO (5 mL). The resulting solutionwas stirred for 24 h at 130° C. After cooling, 10 mL of H₂O was added tothe solution. The resulting solution was extracted with 15 mL of EtOAc,dried over anhydrous Na₂SO₄, and concentrated under vacuum. The residuewas purified by normal phase chromatography on silica gel withEtOAc/petroleum ether (1:1). The collected fractions were concentratedto give 80 mg (25% yield) of the title compound as a white solid. MS:(ES, m/z): 390 [M+H]⁺.

Step-2:N-Hydroxy-2′-oxo-1′-(3-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed a solution of methyl2′-oxo-1′-(3-(trifluoromethyl)phenyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(66 mg, 0.17 mmol, 1 equiv) in THF/MeOH (4:1, 4 mL), NH₂OH (50% in H₂O,1.12 mL, 17 mmol, 100 equiv), and aq. 1N NaOH (0.34 mL, 0.34 mmol, 2equiv). The resulting solution was stirred for 24 h at room temperature.The solids were filtered out. The crude product was purified byPrep-HPLC with the following conditions: Column: XBridge Prep C18 OBD,19×150 mm, 5 μm; Mobile Phase A: water/0.1% formic acid, Mobile Phase B:CH₃CN; Flow rate: 20 mL/min; Gradient: 18% B to 76% B in 8 min;Detector: UV 254 nm, 220 nm. The collected fractions were lyophilized togive 24.2 mg (37% yield) of the title compound as a white solid. ¹H NMR(400 MHz, DMSO-d6) δ (ppm): 10.93 (br s, 1H), 9.00 (br s, 1H), 8.28 (s,1H), 7.89 (d, J=8.4 Hz, 1H), 7.66 (t, J=8.0 Hz, 1H), 7.52 (d, J=8.0 Hz,1H), 7.36-7.34 (m, 2H), 7.27-7.24 (m, 1H), 3.98-3.90 (m, 2H), 3.41 (d,J=16.0 Hz, 1H), 3.30-3.19 (m, 2H), 3.04 (d, J=16.0 Hz, 1H), 2.15 (t,J=6.8 Hz, 2H). MS: (ES, m/z): 391 [M+H]⁺.

TABLE 3-2 Found (ES, m/z) Ex. Structure ¹H-NMR δ (ppm) [M + H]⁺ II-13

(300 MHz, DMSO-d6): 10.9 (br s, 1H), 7.86-7.78 (m, 2H), 7.66-7.56 (m,2H), 7.44-7.19 (m, 3H), 3.80-3.71 (m, 2H), 3.41 (d, J = 22.8 Hz, 1H),3.3-3.17 (m, 2H), 3.04 (d, J = 21.6 Hz, 1H), 2.19 (t, J = 8.8 Hz, 2H)391The following compound was prepared according to the method of Example7-2.

Example 8-2—Preparation of1′-benzyl-N-hydroxy-2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-14)

A solution of methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate in DMA(0.2M, 331 mg, 1.35 mmol) was deprotonated with an equimolar quantity ofNaH (60% dispersion in oil, 54 mg). This solution (150 μL, 30 μmol, 1equiv) was added to a solution of benzyl bromide (0.2M in CH₃CN, 300 μL,60 μmol, 2 equiv) in a 2-dram vial. NaI (18 mg, 120 μmol, 4 equiv) wasadded as a solid in one portion. The vial was sealed and shaken at 50°C. for 24 h, then the solvent was removed under a stream of N₂ (g). Theresidue was diluted with brine (500 μL) and extracted with EtOAc (2×500μL). The combined organic layers were dried under a stream of N₂ (g).THF/MeOH (3:1, 200 μL) was added to the residue. The vial was sealed andshaken at 50° C. for 15 min to dissolve the residue, then cooled to roomtemperature. NH₂OH (50% v/v solution in water, 150 μL) was added,followed by aq. 1N NaOH (100 μL). The mixture was sealed and then shakenat room temperature for 18 h. The reaction mixture was concentratedunder a stream of N₂ (g) at room temperature, then dissolved in 500 L ofDMSO and purified by mass triggered Prep-HPLC using the followingconditions: Column: Waters Sunfire C18, 19×50 mm, 5 μm; Mobile Phase A:water/0.1% formic acid, Mobile Phase B: CH₃CN/0.1% formic acid;Gradient; 15% B up to 100% B in 6 min; Flow rate: 23 mL/min; Detector:UV 254 nm, 220 nm. The product-containing fractions were combined andconcentrated under vacuum to afford 2.0 mg (20% yield) the titlecompound. MS: (ES, m/z): 337 [M+H]⁺.

TABLE 4-2 Found (ES, m/z) Ex. Structure [M + H]⁺ II-15

365 II-16

413 II-17

351 II-18

413 II-19

367 II-20

367 II-21

405 II-22

405 II-23

351 II-24

338 II-25

371 II-26

355 II-27

421 II-28

373 II-29

405 II-30

371 II-31

405 II-32

421 II-33

415 II-34

420 II-35

373 II-36

367 II-37

413 II-38

405 II-39

365 II-40

397 II-41

443 II-42

421 II-43

423 II-44

343 II-45

405 II-46

404 II-47

404 II-48

406 II-49

413 II-50

356 II-51

406 II-52

360 II-53

404 II-54

415 II-55

301The following compounds were prepared according to the parallelsynthesis method of Example 8-2.

Example 9-2—Preparation ofN-hydroxy-1′-(4-(trifluoromethyl)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-56)

Step-1: Methyl2′-thioxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed methyl2′-oxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (1.5 g,6.12 mmol, 1 equiv),2,4-bis(4-methoxyphenyl)-2,4-dithioxo-1,3,2,4-dithiadiphosphetane(Lawesson reagent) (2.37 g, 5.87 mmol, 0.96 equiv), and THF (15 mL). Theresulting solution was stirred for 12 h at room temperature and thenconcentrated under vacuum. The residue was purified by normal phasechromatography on silica gel with EtOAc/petroleum ether (1:3). Thecollected fractions were concentrated to afford 600 mg (38% yield) ofthe title compound as a white solid. MS: (ES, m/z): 262 [M+H]⁺.

Step-2: Methyl 1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 250-mL round-bottom flask was placed methyl2′-thioxo-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (600mg, 2.30 mmol, 1 equiv), NiCl₂.6H₂O (1.9 g, 7.98 mmol, 3.5 equiv), MeOH(30 mL), and THF (20 mL). Then NaBH₄ (400 mg, 10.57 mmol, 4.66 equiv)was added in portions at 0° C. The resulting solution was stirred for 1h at room temperature. The solids were filtered out and the filtrate wasconcentrated under vacuum. The residue was purified by normal phasechromatography on silica gel using MeOH/CH₂Cl₂ (1:10). The collectedfractions were concentrated to afford 400 mg (75% yield) of the titlecompound as white solid. MS: (ES, m/z): 232 [M+H]⁺.

Step-3: Methyl1′-(4-(trifluoromethyl)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed1-(bromomethyl)-4-(trifluoromethyl)benzene (81 mg, 0.34 mmol, 1.1equiv), methyl 1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(69 mg, 0.30 mmol, 1 equiv), THF (10 mL), and a drop of DMF. This wasfollowed by the addition of NaH (30 mg, 1.25 mmol, 2.1 equiv). Theresulting solution was stirred for 12 h at room temperature. Theresulting mixture was concentrated under vacuum. The residue waspurified by normal phase chromatography on silica gel usingEtOAc/petroleum ether (1:1). The collected fractions were concentratedto afford 20 mg (17% yield) of the title compound as colorless oil. MS:(ES, m/z): 390 [M+H]⁺.

Step-4:N-Hydroxy-1′-(4-(trifluoromethyl)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed methyl1′-(4-(trifluoromethyl)benzyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(20 mg, 0.05 mmol, 1 equiv) in THF/MeOH (4:1, 2 mL), NH₂OH (50% in H₂O,0.1 mL, 1.52 mmol, 30 equiv) and aq. 1N NaOH (0.1 mL, 0.1 mmol, 2equiv). The resulting solution was stirred for 2 h at room temperature.The solids were filtered out. The crude product was purified byPrep-HPLC with the following conditions: Column: Gemini-NX C18 110 A,AXIA Packed 150×21.2 mm, 5 μm; Mobile Phase A: water/0.05% TFA, MobilePhase B: CH₃CN; Flow rate: 20 mL/min; Gradient: 10% B to 42% B in 12min; Detector: UV 254 nm, 220 nm. Aqueous 1N HCl (0.05 mL) was added tothe collected fractions and lyophilized to give 3 mg (15% yield) of thetitle compound as the HCl salt as a white solid. ¹H NMR (400 MHz,DMSO-d6) δ (ppm): 10.92 (br s, 1H), 9.01 (br s, 1H), 7.88-7.83 (m, 4H),7.24-7.05 (m, 3H), 4.58-4.45 (m, 2H), 3.55-3.53 (m, 2H), 3.44-3.14 (m,4H), 3.14-2.90 (m, 2H), 2.14-2.00 (m, 1H), 1.98-1.90 (m, 1H). MS: (ES,m/z): 391 [M−HCl+H]⁺.

Example 10-2—Preparation of1′-cyclopropyl-N-hydroxy-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-57)

Step-1: Methyl1′-cyclopropyl-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 50-mL round-bottom flask was placed a solution of methyl1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (180 mg, 0.78mmol, 1 equiv) in CH₂Cl₂ (5 mL), (1-ethoxycyclopropoxy)trimethylsilane(720 mg, 4.13 mmol, 5.3 equiv), AcOH (416 mg, 6.93 mmol, 8.9 equiv), andNaBH₃CN (224 mg, 3.56 mmol, 4.6 equiv). The resulting solution wasstirred overnight at 40° C. The reaction mixture was cooled to roomtemperature and concentrated under vacuum. The crude product waspurified by reverse phase chromatography with the following conditions:Column: C18, 40 g, 20-35 μm; Mobile Phase A: Water/0.05% TFA, MobilePhase B: CH₃CN; Flow rate: 40 mL/min; Gradient: 5% B to 65% B in 30 min.The collected fractions were lyophilized to give 60 mg (28% yield) ofthe title compound as white solid. MS: (ES, m/z): 272 [M+H]⁺.

Step-2:1′-Cyclopropyl-N-hydroxy-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed a solution of1′-cyclopropyl-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(60 mg, 0.22 mmol, 1 equiv) in THF/MeOH (4:1, 3 mL), NH₂OH (50% in H₂O,1.40 mL, 21 mmol, 96 equiv) and aq. 1N NaOH (0.44 mL, 0.44 mmol, 2equiv). The resulting solution was stirred for 4 h at room temperature.The solids were filtered out. The crude product was purified byPrep-HPLC with the following conditions: Column: X Bridge C18, 19×150mm, 5 μm; Mobile Phase A: Water/0.05% TFA, Mobile Phase B: CH₃CN; Flowrate: 20 mL/min; Gradient: 30% B to 70% B in 10 min; Detector: UV 254nm. The collected fractions were lyophilized to give 1.4 mg (2% yield)of the title compound as a white solid. ¹H NMR (300 MHz, DMSO-d6) δ(ppm): 10.80 (br s, 1H), 8.95 (br s, 1H), 7.27-7.23 (m, 2H), 7.16-7.12(m, 1H), 3.07-2.79 (m, 4H), 2.73-2.70 (m, 2H), 2.65-2.57 (m, 2H),1.75-1.65 (m, 3H), 0.35-0.25 (m, 4H). MS: (ES, m/z): 273 [M+H]⁺.

Example 11-2—Preparation ofN-hydroxy-1′-(4-(trifluoromethyl)benzoyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide(II-58)

Step-1: Methyl1′-(4-(trifluoromethyl)benzoyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 50-mL round-bottom flask was placed a solution of methyl1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (30 mg, 0.13mmol, 1 equiv) in CH₂Cl₂ (5 mL), and pyridine (60 mg, 0.76 mmol, 5.85equiv). The reaction mixture was stirred for 30 min at room temperature,and then 4-(trifluoromethyl)benzoyl chloride (45 mg, 0.22 mmol, 1.66equiv) was added. The resulting solution was stirred for 1 h at roomtemperature, then concentrated under vacuum. The residue was purified bynormal phase chromatography on silica gel using EtOAc/petroleum ether(1:2). The collected fractions were concentrated to afford 30 mg (57%yield) of the title compound as colorless oil. MS: (ES, m/z): 404[M+H]⁺.

Step-2:1′-(4-(Trifluoromethyl)benzoyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylicacid

Into a 50-mL round-bottom flask was placed methyl1′-(4-(trifluoromethyl)benzoyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate(30 mg, 0.07 mmol, 1 equiv), aq. 1N NaOH (0.2 mL, 0.2 mmol, 2.8 equiv),and THF (3 mL). The resulting solution was stirred overnight at 80° C.The reaction mixture was cooled to room temperature. The crude productwas purified by reverse phase chromatography with the followingconditions: Column: C18, 40 g, 20-35 μm; Mobile Phase A: Water/0.05%TFA, Mobile Phase B: CH₃CN; Flow rate: 40 mL/min; Gradient: 5% B to 95%B in 35 min; Detector: UV 254 nm. The collected fractions werelyophilized to give 25 mg (86% yield) of the title compound as anoff-white solid. MS: (ES, m/z): 390 [M+H]⁺.

Step-3:N-Hydroxy-1′-(4-(trifluoromethyl)benzoyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed a solution of1′-(4-(trifluoromethyl)benzoyl)-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylicacid (25 mg, 0.06 mmol, 1 equiv) in DMF (3 mL), isopropyl chloroformate(11 mg, 0.10 mmol, 1.54 equiv) and NMM (10 mg, 0.10 mmol, 1.54 equiv).The mixture was stirred for 5 min at room temperature and then NH₂OH.HCl(6 mg, 0.1 mmol, 1.54 equiv) was added. The resulting solution wasstirred overnight at room temperature. The crude product was purified byPrep-HPLC with the following conditions: Column: X Bridge RP, 19×150 mm,5 μm; Mobile Phase A: Water/0.05% formic acid, Mobile Phase B: CH₃CN;Flow rate: 20 mL/min; Gradient: 12% B to 34% B in 9 min; Detector: UV254 nm. The collected fractions were lyophilized to give 4 mg (15%yield) of the title compound as an off-white solid. ¹H NMR (400 MHz,DMSO-d6) δ (ppm): 10.91-10.83 (m, 1H), 9.11 (br s, 1H), 7.83-7.71 (m,4H), 7.35-7.14 (m, 3H), 3.65-3.61 (m, 1H), 3.52-3.49 (m, 2H), 3.38-3.32(m, 1H), 3.16-2.87 (m, 4H), 1.97-1.86 (m, 2H). MS: (ES, m/z): 405[M+H]⁺.

Example 12-2—Preparation of1′-acetyl-N-hydroxy-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide (II-59)

Step-1: Methyl1′-acetyl-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed a solution of methyl1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (70 mg, 0.30mmol, 1 equiv) in CH₂Cl₂ (3 mL), and pyridine (240 mg, 3.03 mmol, 10equiv). The solution was stirred for 20 min at room temperature and thenacetyl chloride (35 mg, 0.45 mmol, 1.47 equiv) was added. The resultingsolution was stirred for 1 h at room temperature and then concentratedunder vacuum. The residue was purified by normal phase chromatography onsilica gel using EtOAc/petroleum ether (1:10). The collected fractionswere concentrated to afford 50 mg (60% yield) of the title compound ascolorless oil. MS: (ES, m/z): 274 [M+H]⁺.

Step-2: 1′-Acetyl-N-hydroxy-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed methyl1′-acetyl-1,3-dihydrospiro[indene-2,3′-pyrrolidine]-4-carboxylate (40mg, 0.15 mmol, 1 equiv) in THF/MeOH (4:1, 4 mL), NH₂OH (50% in H₂O, 0.93mL, 14.07 mmol, 96 equiv), and aq. 1N NaOH (0.29 mL, 0.29 mmol, 2equiv). The resulting solution was stirred for 1 h at room temperature.The solids were filtered out. The crude product was purified byPrep-HPLC with the following conditions: Column: X Bridge RP, 19×150 mm,5 μm; Mobile Phase A: Water/0.05% formic acid, Mobile Phase B: CH₃CN;Flow rate: 20 mL/min; Gradient: 12% B to 34% B in 9 min; Detector: UV254 nm. The collected fractions were lyophilized to give 2.2 mg (5%yield) of the title compound as a brown solid. ¹H NMR (300 MHz, DMSO-d6)δ (ppm): 10.86 (br s, 1H), 7.38-7.17 (m, 3H), 3.56-3.52 (m, 1H),3.41-3.38 (m, 2H), 3.36 (s, 1H), 3.14-3.00 (m, 2H), 2.91-2.85 (m, 2H),1.95-1.82 (m, 5H). MS: (ES, m/z): 275 [M+H]⁺.

Example 13-2—Preparation of1′-acetyl-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide(II-60)

Step-1: 2,2′-(Benzylazanediyl)diethanol

Into a 500-mL round-bottom flask was placed 2,2′-azanediyldiethanol (10g, 95 mmol, 1 equiv), MeCN (140 mL), (bromomethyl)benzene (12 mL), andK₂CO₃ (26.7 g, 193 mmol, 2 equiv). The resulting solution was stirredovernight at 85° C. The reaction mixture was cooled. The solids werefiltered out and the filtrate was concentrated under vacuum. The residuewas diluted with 60 mL of water. The resulting solution was extractedwith 3×60 mL of CH₂Cl₂. The combined organic layers were washed with2×60 mL of water, dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The residue was purified by normal phasechromatography on silica gel using MeOH/CH₂Cl₂ (1:10). The collectedfractions were concentrated under vacuum to afford 7.53 g (41% yield) ofthe title compound as yellow oil. MS: (ES, m/z): 196 [M+H]⁺.

Step-2: N-Benzyl-2-bromo-N-(2-bromoethyl)ethanamine

Into a 500-mL round-bottom flask was placed2,2′-(benzylazanediyl)diethanol (11.3 g, 57.9 mmol, 1 equiv) and CH₂Cl₂(100 mL). This was followed by the dropwise addition of phosphorustribromide (34.6 g, 128 mmol, 2.2 equiv) at 0° C. The resulting solutionwas stirred for 8 h at room temperature. The reaction was then quenchedby the addition of 80 mL of ice-water. The pH value of the solution wasadjusted to 7 with sat. aq. Na₂CO₃ solution. The resulting solution wasextracted with 3×80 mL of CH₂Cl₂. The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Theresidue was purified by normal phase chromatography on silica gel usingCH₂Cl₂/petroleum ether (1:3). The collected fractions were concentratedunder vacuum to afford 3.3 g (18% yield) of the title compound as yellowoil. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.34-7.27 (m, 5H), 3.73 (s, 2H),3.43 (t, J=7.2 Hz, 4H), 2.98 (t, J=7.2 Hz, 1H).

Step-3: 1′-Benzyl-4-bromospiro[indene-2,4′-piperidin]-1 (3H)-one

Into a 500-mL 3-necked round-bottom flask was placed4-bromo-2,3-dihydro-1H-inden-1-one (7.23 g, 34.3 mmol, 1 equiv), DMF (70mL), and N-benzyl-2-bromo-N-(2-bromoethyl)ethanamine (16.5 g, 53.8 mmol,1.57 equiv). This was followed by the addition of NaH (60% dispersion inoil, 5.72 g, 143 mmol, 4.17 equiv), in portions at 0° C. The resultingsolution was stirred for 1 h at 0° C. The reaction mixture was quenchedby the addition of 80 mL of ice-water and the resulting solution wasextracted with 2×100 mL of EtOAc. The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Theresidue was purified by normal phase chromatography on silica gel usingEtOAc/petroleum ether (1:5). The collected fractions were concentratedunder vacuum to afford 4.87 g (38% yield) of the title compound as abrown solid. MS: (ES, m/z): 370 [M+H]⁺.

Step-4: Methyl1′-benzyl-1-oxo-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate

Into a 30-mL pressure tank reactor (40 atm), was placed1′-benzyl-4-bromospiro[indene-2,4′-piperidin]-1(3H)-one (1.15 g, 3.1mmol, 1 equiv), MeOH (15 mL), Pd(dppf)Cl₂ (575 mg, 0.79 mmol, 0.25equiv), and Et₃N (5 mL). CO (g) was introduced into the reactor and theresulting solution was stirred for 2 days at 100° C. The reactionmixture was cooled. The solids were filtered out and the filtrate wasconcentrated under vacuum. The residue was purified by normal phasechromatography on silica gel using EtOAc/petroleum ether (1:1). Thecollected fractions were concentrated under vacuum to afford 518 mg (48%yield) of the title compound as a brown solid. MS: (ES, m/z): 350[M+H]⁺.

Step-5: Methyl1′-benzyl-1-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate

Into a 500-mL round-bottom flask was placed methyl1′-benzyl-1-oxo-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate(2.16 g, 6.2 mmol, 1 equiv) and MeOH (70 mL). This was followed by theaddition of NaBH₄ (707 mg, 18.7 mmol, 3 equiv) in portions at 0° C. Theresulting solution was stirred for 4 h at 0° C. The resulting mixturewas concentrated under vacuum. The residue was purified by normal phasechromatography on silica gel using MeOH/CH₂Cl₂ (1:10). The collectedfractions were concentrated under vacuum to afford 1.85 g (85% yield) ofthe title compound as a brown oil. MS: (ES, m/z): 352 [M+H]⁺.

Step-6: Methyl1′-benzyl-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate

Into a 250-mL round-bottom flask was placed methyl1′-benzyl-1-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate(1.6 g, 4.5 mmol, 1 equiv), TFA (60 mL), and triethylsilane (15 mL). Theresulting solution was stirred for 3 days at room temperature. Theresulting mixture was concentrated under vacuum. The residue waspurified by normal phase chromatography on silica gel using MeOH/CH₂Cl₂(1:10). The collected fractions were concentrated under vacuum to afford1.84 g (crude) of the title compound as a yellow oil. MS: (ES, m/z): 336[M+H]⁺.

Step-7: Methyl 1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylateacetate

Into a 30-mL pressure tank reactor (60 atm), was placed methyl1′-benzyl-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate (1.58g, crude), AcOH (15 mL), and Pd(OH)₂ on carbon (Pearlman's catalyst) (20wt. %, 960 mg). H₂ (g) was introduced into the reactor and the resultingsolution was stirred for 2 days at room temperature. The solids werefiltered out and the filtrate was concentrated under vacuum to afford0.9 g (82% yield over two steps) of the title compound as the acetatesalt as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.38 (br s,2H), 7.86-7.84 (m, 1H), 7.39-7.36 (m, 1H), 7.26-7.23 (m, 1H), 3.97 (s,3H), 3.27-3.14 (m, 6H), 2.91-2.83 (m, 2H), 1.92-1.90 (m, 4H). MS: (ES,m/z): 246 [M+H]⁺.

Step-8: Methyl1′-acetyl-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed methyl1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate acetate (40 mg,0.13 mmol, 1 equiv), CH₂Cl₂ (3 mL), pyridine (64.5 mg, 0.82 mmol, 6.3equiv), and acetyl chloride (28 mg, 0.36 mmol, 2.8 equiv). The resultingsolution was stirred for 2 h at room temperature. The resulting mixturewas concentrated under vacuum. The residue was purified by reverse phasechromatography with the following conditions: Column: C18, 40 g, 20-35μm; Mobile Phase A: Water/0.05% TFA, Mobile Phase B: CH₃CN; Flow rate:40 mL/min; Gradient: 5% B to 50% B in 25 min; Detector: UV 254 nm. Thecollected fractions were lyophilized to afford 25.7 mg (68% yield) ofthe title compound as a colorless oil. MS: (ES, m/z): 288 [M+H]⁺.

Step-9:1′-Acetyl-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed methyl1′-acetyl-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate (25.7mg, 0.09 mmol, 1 equiv), THF/MeOH (4:1, 2 mL), NH₂OH (50% in H₂O, 591mg, 8.95 mmol, 100 equiv), and aq. 1N NaOH (0.18 mL, 0.18 mmol, 2equiv). The resulting solution was stirred for 7 h at room temperature.The crude product was purified by Prep-HPLC with the followingconditions: Column: XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile PhaseA: water/0.05% formic acid, Mobile Phase B: CH₃CN; Detector: UV 254 nm,220 nm. The collected fractions were lyophilized to give 9 mg (35%yield) of the title compound as a light brown solid. ¹H NMR (400 MHz,DMSO-d6) δ (ppm): 10.91-10.83 (m, 1H), 7.32-7.21 (m, 2H), 7.21-7.15 (m,1H), 3.61-3.37 (m, 4H), 3.02-2.93 (m, 2H), 2.85-2.79 (m, 2H), 2.00 (s,3H), 1.57-1.50 (m, 2H), 1.49-1.38 (m, 2H). MS: (ES, m/z): 289 [M+H]⁺.

Example 14-2—Preparation of1′-(4-chlorobenzoyl)-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide(II-61)

Step-1: Methyl1′-(4-chlorobenzoyl)-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed 4-chlorobenzoic acid (95.5mg, 0.61 mmol, 1.85 equiv), DMF (3 mL), EDC (130.7 mg, 0.84 mmol, 2.55equiv), HOBt (55.2 mg, 0.41 mmol, 1.24 equiv), Et₃N (136.3 mg, 1.35mmol, 4.09 equiv), and methyl1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate acetate (100 mg,0.33 mmol, 1 equiv). The resulting solution was stirred overnight atroom temperature. The resulting solution was diluted with 10 mL of waterand extracted with 3×10 mL of EtOAc. The combined organic layers weredried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum.The residue was purified by normal phase chromatography on silica gelusing EtOAc/petroleum ether (1:5). The collected fractions wereconcentrated under vacuum to afford 58.2 mg (46% yield) of the titlecompound as a yellow oil. MS: (ES, m/z): 384 [M+H]⁺.

Step-2:1′-(4-Chlorobenzoyl)-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed methyl1′-(4-chlorobenzoyl)-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate(58.2 mg, 0.15 mmol, 1 equiv), THF/MeOH (4:1, 2 mL), NH₂OH (50% in H₂O,1003 mg, 15 mmol, 100 equiv), and aq. 1N NaOH (0.3 mL, 0.30 mmol, 2equiv). The resulting solution was stirred for 5 h at room temperature.The crude product was purified by Prep-HPLC with the followingconditions: Column: XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile PhaseA: water/0.05% TFA, Mobile Phase B: CH₃CN; Detector: UV 254 nm. Thecollected fractions were lyophilized to give 17.3 mg (30% yield) of thetitle compound as a pink solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm):10.91-10.81 (m, 1H), 7.52-7.50 (m, 2H), 7.49-7.43 (m, 2H), 7.30-7.18 (m,3H), 3.79-3.51 (m, 2H), 3.41-3.25 (m, 2H), 2.99 (s, 2H), 2.84 (s, 2H),1.69-1.42 (m, 4H). MS: (ES, m/z): 385 [M+H]⁺.

Example 15-2—Preparation of1′-(4-chlorobenzyl)-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide(II-62)

Step-1: Methyl1′-(4-chlorobenzyl)-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate

Into a 25-mL round-bottom flask was placed methyl1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate acetate (100 mg,0.33 mmol, 1 equiv), CH₂Cl₂ (3 mL), and 4-chlorobenzaldehyde (60 mg,0.43 mmol, 1.3 equiv). The resulting solution was stirred for 1 h atroom temperature. Then NaBH(OAc)₃ (129.8 mg, 0.61 mmol, 1.85 equiv) wasadded. The resulting solution was allowed to react, with stirring,overnight at room temperature. The reaction mixture was then quenchedwith 20 mL of water and extracted with 3×20 mL of CH₂Cl₂. The combinedorganic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The residue was purified by normal phasechromatography on silica gel using EtOAc/petroleum ether (1:5). Thecollected fractions were concentrated under vacuum to afford 60 mg (50%yield) of the title compound as a white solid. MS: (ES, m/z): 370[M+H]⁺.

Step-2:1′-(4-Chlorobenzyl)-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide

Into a 25-mL round-bottom flask was placed methyl1′-(4-chlorobenzyl)-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate(60 mg, 0.16 mmol, 1 equiv), THF/MeOH (4:1, 2 mL), NH₂OH (50% in H₂O,1073 mg, 16 mmol, 100 equiv), and aq. 1N NaOH (0.8 mL, 0.8 mmol, 5equiv). The resulting solution was stirred for 8 h at room temperature.The crude product was purified by Prep-HPLC with the followingconditions: Column: XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile PhaseA: water/0.05% TFA, Mobile Phase B: CH₃CN; Detector: UV 254 nm. Thecollected fractions were lyophilized to give 13.5 mg (20% yield) of thetitle compound as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm):10.91-10.88 (d, J=12 Hz, 1H), 10.44-10.38 (m, 1H), 9.05-8.69 (m, 1H),7.68-7.62 (m, 2H), 7.56-7.52 (m, 2H), 7.32-7.25 (m, 2H), 7.21-7.17 (m,1H), 4.34-4.29 (m, 2H), 3.25-3.17 (m, 2H), 3.12-3.00 (m, 3H), 2.93 (d,J=12 Hz, 2H), 2.78 (s, 1H), 1.93-1.84 (m, 2H), 1.78-1.66 (m, 2H). MS:(ES, m/z): 371 [M+H]⁺.

Example 16-2—Preparation of1′-cyclopropyl-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide(II-63)

Step-1: Methyl1′-cyclopropyl-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate

Into an 8-mL round-bottom flask was placed methyl1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate acetate (100 mg,0.33 mmol, 1 equiv), (1-ethoxycyclopropoxy)trimethylsilane (430 mg, 2.47mmol, 7.48 equiv), CH₂Cl₂ (5 mL), and AcOH (240 mg, 4.00 mmol, 12.12equiv). The resulting solution was stirred for 30 min at roomtemperature. Then NaBH₃CN (130 mg, 2.07 mmol, 6.27 equiv) was added. Theresulting solution was allowed to react, with stirring, overnight at 40°C. The reaction mixture was then cooled to room temperature, dilutedwith 20 mL of water, and extracted with 3×20 mL of CH₂Cl₂. The combinedorganic layers were dried with anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The residue was purified by normal phasechromatography on silica gel using MeOH/CH₂Cl₂ (1:5). The collectedfractions were concentrated under vacuum to afford 50 mg (crude) of thetitle compound as yellow oil. MS: (ES, m/z): 286 [M+H]⁺.

Step-2:1′-Cyclopropyl-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide

Into a 100-mL round-bottom flask was placed methyl1′-cyclopropyl-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate(66 mg, 0.23 mmol, 1 equiv), THF/MeOH (4:1, 2 mL), NH₂OH (50% in H₂O,1.53 g, 23 mmol, 100 equiv), and aq. 1N NaOH (0.46 mL, 0.46 mmol, 2equiv). The resulting solution was stirred overnight at roomtemperature. The crude product was purified by Prep-HPLC with thefollowing conditions: Column: XBridge Prep C18 OBD, 19×250 mm, 5 μm;Mobile Phase A: water/0.05% TFA, Mobile Phase B: CH₃CN; Gradient: 5% Bto 23% B in 9 min; Detector: UV 254 nm, 220 nm. The collected fractionswere concentrated under vacuum to afford 12.2 mg (18% yield) of thetitle compound as a colorless oil. ¹H NMR (400 MHz, DMSO-d6) δ (ppm):10.88 (br s, 1H), 10.32 (m, 1H), 7.37-7.11 (m, 3H), 3.38-3.26 (m, 2H),3.24-3.22 (m, 2H), 3.08 (s, 1H), 2.98-2.76 (m, 4H), 1.98-1.91 (m, 2H),1.72-1.67 (m, 2H), 1.10 (s, 2H), 0.77 (s 2H). MS: (ES, m/z): 287 [M+H]⁺.

Example 17-2—Preparation of1′-benzyl-N-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide(II-64)

Into a 25-mL round-bottom flask was placed methyl1′-benzyl-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate (50 mg,0.15 mmol, 1 equiv), THF/MeOH (4:1, 2 mL), NH₂OH (50% in H₂O, 985 mg, 15mmol, 100 equiv), and aq. 1N NaOH (0.3 mL, 0.30 mmol, 2 equiv). Theresulting solution was stirred for 8.5 h at room temperature. The crudeproduct was purified by Prep-HPLC with the following conditions: Column:XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile Phase A: water/0.05% TFA,Mobile Phase B: CH₃CN; Detector: UV 254 nm, 220 nm. The collectedfractions were concentrated under vacuum to afford 6.4 mg (12% yield) ofthe title compound as a as a pink solid. ¹H NMR (400 MHz, DMSO-d6) δ(ppm): 10.88-10.87 (m, 1H), 10.36-10.29 (m, 1H), 9.15-8.82 (m, 1H),7.63-7.58 (m, 2H), 7.47 (s, 3H), 7.32-7.24 (m, 2H), 7.22-7.18 (m, 1H),4.35-4.31 (m, 2H), 3.22-3.19 (m, 2H), 3.12-3.02 (m, 3H), 2.94-2.91 (m,2H), 2.78 (s, 1H), 1.94-1.88 (m, 2H), 1.75-1.70 (m, 2H). MS: (ES, m/z):337 [M+H]⁺.

Example 18-2—Preparation of1′-benzyl-N-hydroxy-1-oxo-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide(II-65)

Into a 25-mL round-bottom flask was placed methyl1′-benzyl-1-oxo-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate(50 mg, 0.14 mmol, 1 equiv), THF/MeOH (4:1, 2 mL), NH₂OH (50% in H₂O,946 mg, 14 mmol, 100 equiv), and aq. 1N NaOH (0.29 mL, 0.29 mmol, 2equiv). The resulting solution was stirred for 2 h at room temperature.The crude product was purified by Prep-HPLC with the followingconditions: Column: XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile PhaseA: water/0.05% TFA, Mobile Phase B: CH₃CN; Detector: UV 254 nm. Thecollected fractions were concentrated under vacuum to afford 3.6 mg (7%yield) of the title compound as a as a pink solid. ¹H NMR (400 MHz,DMSO-d6) δ (ppm): 11.19 (s, 1H), 10.02 (br s, 1H), 9.18 (s, 1H),7.86-7.63 (m, 2H), 7.58-7.48 (m, 6H), 4.39 (s, 2H), 3.39-3.34 (m, 4H),3.20-3.11 (m, 2H), 2.11-1.95 (m, 2H), 1.66-1.63 (m, 2H). MS: (ES, m/z):351 [M+H]⁺.

Example 19-2—Preparation of 1′-benzyl-N,1-dihydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxamide(II-66)

Into a 25-mL round-bottom flask was placed methyl1′-benzyl-1-hydroxy-1,3-dihydrospiro[indene-2,4′-piperidine]-4-carboxylate(50 mg, 0.14 mmol, 1 equiv), THF/MeOH (4:1, 2 mL), NH₂OH (50% in H₂O,940 mg, 14 mmol, 100 equiv), and aq. 1N NaOH (0.28 mL, 0.28 mmol, 2equiv). The resulting solution was stirred for 4 h at room temperature.The crude product was purified by Prep-HPLC with the followingconditions: Column: XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile PhaseA: water/0.1% formic acid, Mobile Phase B: CH₃CN; Detector: UV 254 nm,220 nm. The collected fractions were concentrated under vacuum to afford14.5 mg (26% yield) of the title compound as a pink solid. ¹H NMR (400MHz, DMSO-d6) δ (ppm): 10.95 (s, 1H), 10.42-10.01 (m, 1H), 8.99 (s, 1H),7.63-7.09 (m, 8H), 5.65-5.44 (m, 1H), 4.83-4.59 (m, 1H), 4.33 (s, 2H),3.29-2.73 (m, 6H), 2.11-1.22 (m, 4H). MS: (ES, m/z): 353 [M+H]⁺.

Example 20-2—Preparation of1′-(4-chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide(II-67)

Step-1: 1-Bromo-2,3-bis(bromomethyl)benzene

Into a 250-mL 3-necked round-bottom flask was placed1-bromo-2,3-dimethylbenzene (10 g, 54 mmol, 1 equiv), carbontetrachloride (60 mL), N-bromosuccinimide (38 g, 213.5 mmol, 3.95equiv), AIBN (253 mg, 1.54 mmol, 0.03 equiv). The resulting solution wasstirred for 15 h at 85° C. in an oil bath. The reaction mixture wascooled to room temperature. The solids were filtered out and thefiltrate was washed with 2×200 mL of water and 200 mL of brine, driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give22 g (crude) of the title compound as a brown oil. GCMS: (ES, m/z): 340.

Step-2: Mixture of methyl5-bromo-1,2,3,4-tetrahydronaphthalene-2-carboxylate and methyl8-bromo-1,2,3,4-tetrahydronaphthalene-2-carboxylate

Into a 500-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen was placed 1-bromo-2,3-bis(bromomethyl)benzene(22 g, 64 mmol, 1 equiv), DMF (100 mL), methyl prop-2-enoate (27.5 g,319 mmol, 5 equiv), and sodium iodide (38.4 g, 256 mmol, 4 equiv). Theresulting solution was stirred for 18 h at 90° C. in an oil bath. Thereaction mixture was cooled to room temperature and then quenched by theaddition of 100 mL of water. The resulting solution was decolorized bythe addition of aq. 5% Na₂S₂O₃ solution and extracted with 3×150 mL ofEtOAc. The combined organic layers were washed with 200 mL of brine,dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum.The residue was purified by normal phase chromatography on silica gelusing EtOAc/petroleum ether (1:20). The collected fractions wereconcentrated under vacuum to afford 10.5 g (61% yield) of the titlecompounds as a light brown oil. GCMS: (ES, m/z): 268.

Step-3: Mixture of dimethyl 5,6,7,8-tetrahydronaphthalene-1,6-dicarboxylate and dimethyl 5,6,7,8-tetrahydronaphthalene-1,7-dicarboxylate

Into a 50-mL pressure tank reactor (60 atm), was placed the mixture ofmethyl 5-bromo-1,2,3,4-tetrahydronaphthalene-2-carboxylate and methyl8-bromo-1,2,3,4-tetrahydronaphthalene-2-carboxylate (6.95 g, 25.84 mmol,1 equiv), MeOH (20 mL), Pd(dppf)Cl₂ (2.30 g, 3.14 mmol, 0.12 equiv), andEt₃N (10.44 g, 103.17 mmol, 4.00 equiv). CO (g) was introduced and theresulting solution was stirred for 24 h at 120° C. The resulting mixturewas cooled to room temperature and concentrated under vacuum. Theresidue was purified by normal phase chromatography on silica gel usingEtOAc/petroleum ether (1:10). The collected fractions were concentratedunder vacuum to afford 3.14 g (49% yield) of the title compounds as agreenish oil. MS: (ES, m/z): 249 [M+H]⁺.

Step-4: Mixture of dimethyl6-(cyanomethyl)-5,6,7,8-tetrahydronaphthalene-1,6-dicarboxylate anddimethyl 7-(cyanomethyl)-5,6,7,8-tetrahydronaphthalene-1,7-dicarboxylate

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed the mixture of dimethyl5,6,7,8-tetrahydronaphthalene-1,6-dicarboxylate and dimethyl5,6,7,8-tetrahydronaphthalene-1,7-dicarboxylate (3.14 g, 12.66 mmol, 1equiv) in THF (25 mL). This was followed by the addition of lithiumdiisopropylamide solution (2M in THF, 12.6 mL, 25.32 mmol, 2 equiv)dropwise with stirring at −78° C. and then the mixture was stirred for 1hr at −78° C. To this was added 2-bromoacetonitrile (4.56 g, 38.02 mmol,3 equiv) dropwise with stirring at −78° C. The resulting solution wasstirred for 3 h at −78° C. The reaction mixture was then quenched by theaddition of 5 mL of aq. sat. NH₄Cl solution and slowly warmed to roomtemperature. The resulting mixture was poured into 50 mL of water andextracted with 3×50 mL of EtOAc. The combined organic layers were washedwith 100 mL of brine, dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The residue was purified by normal phasechromatography on silica gel using EtOAc/petroleum ether (1:5). Thecollected fractions were concentrated under vacuum to give 2.22 g (61%yield) of the title compound as a brown oil. MS: (ES, m/z): 288 [M+H]⁺.

Step-5: Methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxylateand Methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate

Into a 250-mL round-bottom flask was placed the mixture of dimethyl6-(cyanomethyl)-5,6,7,8-tetrahydronaphthalene-1,6-dicarboxylate anddimethyl 7-(cyanomethyl)-5,6,7,8-tetrahydronaphthalene-1,7-dicarboxylate(2.22 g, 7.74 mmol, 1 equiv), MeOH (60 mL), AcOH (14 mL), and PtO₂ (2 g,8.81 mmol, 1.14 equiv). H₂ (g) was introduced and the resulting solutionwas stirred for 3.5 h at room temperature. The solids were filtered outand the filtrate was concentrated under vacuum. The residue wasdissolved in 20 mL of NH₃ solution (7M in MeOH) and stirred for 18 h atroom temperature. The resulting mixture was concentrated under vacuum.The crude product was purified by reversed phase chromatography with thefollowing conditions: Column: C18, 120 g, 20-45 μm, 100 A; Mobile PhaseA: Water/0.1% formic acid, Mobile Phase B: CH₃CN; Gradient: 15% B to 45%B in 50 min; Detector: UV 254 nm. The first eluting isomer was collectedand concentrated under vacuum to give 100 mg (7% yield) of the titlecompound methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxylateas a white solid. Structure assignment based on NOESY, HSQC, and HMBCNMR experiments: ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 7.68 (s, 1H, NH),7.61 (d, 1H, CH²), 7.32 (d, 1H, CH⁴), 7.22 (t, 1H, CH³), 3.81 (s, 3H,CH₃), 3.19-3.22 (m, 2H, CH⁹), 2.92-3.18 (m, 2H, CH⁷), 2.76 (dd, 2H,CH⁵), 1.70-1.95 (m, 4H, CH⁶ and CH¹⁰). ¹³C NMR (100 MHz, DMSO-d6) δ(ppm): 180.1, 167.6, 136.22, 136.18, 133.5, 129.6, 127.7, 125.4, 51.9,41.1, 37.9, 36.2 (C⁵), 31.1, 28.5, 23.9 (C⁷). NOESY correlation observedbetween H⁴ and H⁵. HMBC correlation observed between H⁴ and C⁵. MS: (ES,m/z): 260 [M+H]⁺.

The second eluting isomer was collected and concentrated under vacuum togive 800 mg (53% yield) of the title compound methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylateas a light yellow solid. Structure assignment based on NOESY, HSQC, andHMBC NMR experiments: ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 7.68 (s, 1H,NH), 7.60 (d, 1H, CH^(b)), 7.34 (d, 1H, CH^(d)), 7.23 (app t, 1H,CF^(c)), 3.80 (s, 3H, CH₃), 3.17-3.21 (m, 2H, CH^(i)), 2.87-2.94 (m, 2H,CH^(g)), 2.80-2.85 (m, 2H, CH^(e)), 1.61-1.93 (m, 4H, CH^(f) andCH^(j)). ¹³C NMR (100 MHz, DMSO-d6) δ (ppm): 180.3, 167.6, 136.8, 135.5,132.7, 130.4, 127.6, 125.4, 51.9, 41.6, 37.9, 34.1 (C^(g)), 31.5, 27.9,25.8 (C^(e)). NOESY correlation observed between H^(d) and H^(e). HMBCcorrelation observed between H^(d) and C^(e). MS: (ES, m/z): 260 [M+H]⁺.

Step-6: Methyl1′-(4-chloro-3-(trifluoromethyl)benzyl)-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate

Into an 8-mL vial was placed methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(50 mg, 0.19 mmol, 1 equiv), DMF (3 mL). This was followed by theaddition of NaH (60% dispersion in oil, 23 mg, 0.58 mmol, 3 equiv), inportions at 0° C. The mixture was stirred for 30 min at roomtemperature. To this was added a solution of4-(bromomethyl)-1-chloro-2-(trifluoromethyl)benzene (158 mg, 0.58 mmol,3 equiv) in DMF (0.5 mL) dropwise with stirring at 0° C. The resultingsolution was stirred for 2 h at room temperature. The reaction mixturewas then poured into 20 mL of ice-water and extracted with 3×20 mL ofEtOAc. The combined organic layers were washed with 30 mL of brine,dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum.The residue was purified by normal phase chromatography on silica gelwith EtOAc/petroleum ether (1:3). The collected fractions wereconcentrated under vacuum to give 100 mg (crude) of the title compoundas light yellow oil. MS: (ES, m/z): 452 [M+H]⁺.

Step-7:1′-(4-Chloro-3-(trifluoromethyl)benzyl)-N-hydroxy-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide

Into a 25-mL round-bottom flask was placed methyl1′-(4-chloro-3-(trifluoromethyl)benzyl)-2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(100 mg, 0.13 mmol, 1 equiv), THF/MeOH (4:1, 3 mL), NH₂OH (50% in H₂O,0.26 mL, 4 mmol, 30 equiv), and aq. 1N NaOH (0.26 mL, 0.26 mmol, 2equiv). The resulting solution was stirred for 4 h at room temperature.The crude product was purified by Prep-HPLC with the followingconditions: Column: XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile PhaseA: water/0.1% formic acid, Mobile Phase B: CH₃CN; Gradient: 35% B to 50%B in 8 min, up to 52% B in 1 min; Detector: UV 254 nm, 220 nm. Thecollected fractions were lyophilized to give 20.5 mg (20% yield) of thetitle compound as an off-white solid. ¹H NMR (300 MHz, DMSO-d6) δ (ppm):10.79 (br s, 1H), 9.03 (br s, 1H), 7.76-14 (m, 2H), 7.76-7.74 (m, 2H),7.55-7.52 (m, 1H), 7.22-7.07 (m, 3H), 4.60 (d, J=15.3 Hz, 1H), 4.43 (d,J=15.3 Hz, 1H), 3.27-3.22 (m, 2H), 2.98-2.88 (m, 3H), 2.66 (d, J=17.4Hz, 1H), 1.93-1.81 (m, 2H), 1.70-1.63 (m, 2H). MS: (ES, m/z): 453 [M+H]⁺

TABLE 5-2 Found (ES, m/z) Ex. Structure ¹H-NMIR δ (ppm) [M + H]⁺ II-68

(400 MHz, DMSO-d6): 10.79 (br s, 1H), 9.01 (br s, 1H), 7.82-7.78 (m,1H), 7.37 (d, J = 12.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.21-7.14 (m,2H), 7.08 (d, J = 6.4 Hz, 1H), 4.60-4.44 (m, 2H), 3.27-3.21 (m, 2H),2.97-2.84 (m, 3H), 2.69-2.64 (m, 1H), 1.98-1.92 (m, 1H), 1.87-1.82 (m,1H), 1.77-1.59 (m, 2H) 437 II-69

(400 MHz, DMSO-d6): 10.80 (br s, 1H), 9.08 (br s, 1H), 7.43 (d, J = 8.4Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.25- 7.13 (m, 2H), 7.09-7.07 (m,1H), 4.47- 4.35 (m, 2H), 3.23-3.16 (m, 2H), 2.95- 2.83 (m, 3H),2.67-2.61 (m, 1H), 1.94- 1.79 (m, 2H), 1.68-1.61 (m, 2H) 385The following compounds were prepared according to the method of Example20-2.

TABLE 6-2 Found (ES, m/z) Ex. Structure ¹H-NMR δ (ppm) [M + H]⁺ II-84

(300 MHz, DMSO-d6): 10.80 (br s, 1H), 9.03 (br s, 1H), 7.77-7.73 (m,2H), 7.57-7.54 (m, 1H), 7.19-7.08 (m, 3H), 4.51 (s, 1H), 3.28-3.24 (m,2H), 2.95- 2.81 (m, 3H), 2.68-2.63 (m, 1H), 1.97- 1.69 (m, 4H) 453 II-85

(400 MHz, DMSO-d6): 11.79 (br s, 1H), 9.02 (br s, 1H), 7.73 (d, J = 8.4Hz, 2H), 7.45 (d, J = 8 Hz, 2H), 7.17- 7.06 (m, 3H), 4.51 (s, 2H),3.25-3.22 (m, 2H), 2.93-2.79 (m, 3H), 2.67-2.65 (m, 1H), 1.94-1.87 (m,1H), 1.84-1.76 (m, 1H), 1.73-1.65 (m, 2H) 419 II-86

(300 MHz, DMSO-d6): 10.78 (br s, 1H), 9.03 (br s, 1H), 7.83-7.77 (m,1H), 7.38-7.27 (m, 2H), 7.20-7.07 (m, 3H), 4.53 (2, 3H), 3.29-3.27 (m,2H), 2.95- 2.81 (m, 3H), 2.68 (d, J = 16.2 Hz, 1H), 1.99-1.89 (m, 1H),1.86-1.69 (m, 3H) 437 II-87

(300 MHz, DMSO-d6): 10.77 (br s, 1H), 9.04 (br s, 1H), 7.43 (d, J = 8.1Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 7.17- 7.07 (m, 3H), 4.41 (s, 2H),3.24-3.19 (m, 2H), 2.95-2.85 (m, 3H), 2.63 (d, J = 16.8 Hz, 1H),1.91-1.67 (m, 4H) 385The following compounds were prepared according to the method of Example20-2, with the following modification: In Step 6, methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxylatewas used.

Example 21-2—Preparation ofN-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)benzyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide(II-70)

Step-1: Methyl2′-oxo-1′-(4-(trifluoromethyl)benzyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate

Into an 8-mL vial was placed methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(150 mg, 0.58 mmol, 1 equiv) and DMF (4 mL). This was followed by theaddition of NaH (60% dispersion in oil, 69 mg, 1.74 mmol, 3 equiv), inportions at 0° C. The resulting mixture was stirred for 30 min at roomtemperature. To this was added1-(bromomethyl)-4-(trifluoromethyl)benzene (276 mg, 1.16 mmol, 2 equiv)dropwise with stirring at 0° C. The resulting solution was stirred for 2h at room temperature. The reaction mixture was then poured into 20 mLof aq. sat. NH₄Cl solution. The resulting solution was extracted with3×20 mL of EtOAc. The combined organic layers were dried over anhydrousNa₂SO₄, filtered, and concentrated under vacuum. The residue waspurified by normal phase chromatography on silica gel usingEtOAc/petroleum ether (1:2). The collected fraction was concentratedunder vacuum to give 76 mg (31% yield) of the title compound as lightyellow oil. MS: (ES, m/z): 418 [M+H]⁺.

Step-2:2′-Oxo-1′-(4-(trifluoromethyl)benzyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylicacid

Into a 25-mL round-bottom flask was placed methyl2′-oxo-1′-(4-(trifluoromethyl)benzyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(76 mg, 0.18 mmol, 1 equiv) and THF (3 mL). This was followed by theaddition of LiOH (40 mg, 1.67 mmol, 9 equiv) in water (3 mL) dropwisewith stirring at 0° C. The resulting solution was stirred for 20 h atroom temperature. The pH value of the solution was adjusted to 3 with 2NHCl and the resulting solution was extracted with 3×20 mL of EtOAc. Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The collected fraction was concentrated undervacuum to give 29 mg (39% yield) of the title compound as off-whitesolid. MS: (ES, m/z): 404 [M+H]⁺.

Step-3:N-Hydroxy-2′-oxo-1′-(4-(trifluoromethyl)benzyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide

Into an 8-mL vial was placed2′-oxo-1′-(4-(trifluoromethyl)benzyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylicacid (29 mg, 0.07 mmol, 1 equiv) and DMA (3 mL). This was followed bythe addition of isopropyl chloroformate (88 mg, 0.72 mmol, 10 equiv)dropwise with stirring at 0° C. To this was added NMM (73 mg, 0.72 mmol,10 equiv) dropwise with stirring at 0° C. The mixture was stirring for 5min at 0° C. To the mixture was added a solution of NH₂OH.HCl (50 mg,0.72 mmol, 10 equiv) in DMA (1 mL) dropwise with stirring at 0° C. Theresulting solution was stirred for 24 h at room temperature. The crudeproduct was purified by Prep-HPLC with the following conditions: Column:XBridge Prep C18 OBD, 19×150 mm, 5 μm; Mobile Phase A: water/0.1% formicacid, Mobile Phase B: CH₃CN; Gradient: 25% B to 39% B in 7 min, hold at39% B for 5 min; Detector: UV 254 nm, 220 nm. The collected fractionswere lyophilized to give 8.3 mg (27% yield) of the title compound as anoff-white solid. ¹H NMR (300 MHz, DMSO-d6) δ (ppm): 10.80 (br s, 1H),9.03 (br s, 1H), 7.74 (d, J=8.1 Hz, 2H), 7.45 (d, J=7.8 Hz, 2H),7.21-7.07 (m, 3H), 4.59-4.44 (m, 2H), 3.23-3.21 (m, 2H), 2.98-2.83 (m,3H), 2.72-2.63 (m, 1H), 1.97-1.79 (m, 2H), 1.69-1.64 (m, 2H). MS: (ES,m/z): 419 [M+H]⁺.

Example 22-2—Preparation ofN-hydroxy-2′-oxo-1′-(4-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide(II-71)

Step-1: Methyl2′-oxo-1′-(4-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate

Into a 50-mL round-bottom flask was placed methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(50 mg, 0.19 mmol, 1 equiv), CH₂Cl₂ (10 mL),[4-(trifluoromethyl)phenyl]boronic acid (72.2 mg, 0.38 mmol, 2 equiv),Cu(OAc)₂ (69.2 mg, 0.38 mmol, 2 equiv), pyridine (22.5 mg, 0.28 mmol,1.5 equiv), Et₃N (57.6 mg, 0.57 mmol, 3 equiv), and 4 Å molecular sieves(100 mg). O₂ (g) was introduced and the resulting solution was stirredfor 36 h at 60° C. The reaction mixture was then cooled to roomtemperature. The solids were filtered out and the filtrate wasconcentrated under vacuum. The residue was purified by prep-TLC withEtOAc/petroleum ether (1:3). The collected fractions were concentratedunder vacuum to give 30 mg (38% yield) of the title compound as a whitesolid. MS: (ES, m/z): 404 [M+H]⁺.

Step-2:N-Hydroxy-2′-oxo-1′-(4-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide

Into a 25-mL round-bottom flask was placed methyl2′-oxo-1′-(4-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(50 mg, 0.12 mmol, 1 equiv), THF/MeOH (4:1, 3 mL), NH₂OH (50% in H₂O,491 mg, 7.44 mmol, 60 equiv). This was followed by the addition of aq.1N NaOH (0.25 mL, 0.25 mmol, 2 equiv) dropwise with stirring. Theresulting solution was stirred for 2 h at room temperature. The solidswere filtered out. The crude product was purified by Prep-HPLC with thefollowing conditions: Column: XBridge Prep C18 OBD, 19×150 mm, 5 μm;Mobile Phase A: water/0.05% NH₃.H₂O, Mobile Phase B: CH₃CN; Gradient:27% B to 48% B in 7 min, hold at 48% B for 2 min; Detector: UV 254 nm,220 nm. The collected fractions were lyophilized to give 4.2 mg (8%yield) of the title compound as a white solid. ¹H NMR (300 MHz, DMSO-d6)δ (ppm): 7.97 (d, J=8.7 Hz, 2H), 7.77 (d, J=8.7 Hz, 2H), 7.20-7.13 (m,3H), 3.92-3.83 (m, 2H), 2.92-2.76 (m, 4H), 2.08-1.75 (m, 4H), MS: (ES,m/z): 405 [M+H]⁺.

TABLE 7-2 Found (ES, m/z) Ex. Structure ¹H-NMR δ (ppm) [M + H]⁺ II-88

(300 MHz, DMSO-d6): 10.79 (br s, 1H), 9.03 (br s, 1H), 7.95 (d, J = 8.7Hz, 2H), 7.76 (d, J = 9 Hz, 2H), 7.21- 7.09 (m, 3H), 3.94-3.89 (m, 2H),2.99- 2.78 (m, 4H), 2.11-2.05 (m, 1H), 1.92- 1.85 (m, 3H) 405 II-89

(300 MHz, DMSO-d6): 10.80 (br s, 1H), 9.04 (br s, 1H), 8.27 (s, 1H),7.87 (d, J = 8.7 Hz, 1H), 7.67-7.62 (m, 1H), 7.50 (d, J = 7.8 Hz, 1H),7.19-7.09 (m, 3H), 3.95-3.90 (m, 2H), 2.99-2.72 (m, 4H), 2.11-2.02 (m,1H), 1.91-1.83 (m, 3H) 405The following compounds were prepared according to the method of Example22-2, with the following modification: In Step 1, methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxylatewas used.

Example 23-2—Preparation ofN-hydroxy-2′-oxo-1′-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide(II-72)

Step-1: Methyl2′-oxo-1′-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate

Into a 50-mL round-bottom flask was placed methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(50 mg, 0.19 mmol, 1 equiv), THF (10 mL),[3-(trifluoromethyl)phenyl]boronic acid (183.116 mg, 0.96 mmol, 5equiv), Cu(OAc)₂ (35 mg, 0.19 mmol, 1 equiv), Et₃N (59 mg, 0.58 mmol, 3equiv), pyridine (22.879 mg, 0.29 mmol, 1.5 equiv) and 4 Å molecularsieves (100 mg). O₂ (g) was introduced and the resulting solution wasstirred for 18 h at 60° C. in an oil bath. The solids were filtered out.The resulting mixture was concentrated under vacuum. The residue waspurified by normal phase chromatography on silica gel usingEtOAc/petroleum ether (2:3). The collected fractions were concentratedunder vacuum to give 32 mg (41% yield) of the title compound as lightbrown oil. MS: (ES, m/z): 404 [M+H]⁺.

Step-2:2′-Oxo-1′-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylicacid

Into a 25-mL round-bottom flask was placed methyl2′-oxo-1′-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(32 mg, 0.08 mmol, 1 equiv) and THF (5 mL). This was followed by theaddition of a solution of LiOH (19 mg, 0.79 mmol, 10 equiv) in water (4mL) dropwise with stirring at 0° C. The resulting solution was stirredfor 1.5 days at room temperature. The pH value of the solution wasadjusted to 3 with 2N HCl at 0° C. and was extracted with 3×15 mL ofEtOAc The combined organic layers were dried over anhydrous Na₂SO₄,filtered, and concentrated under vacuum. The collected fraction wasconcentrated under vacuum to give 30 mg (85% yield) of the titlecompound as a light yellow solid. MS: (ES, m/z): 390 [M+H]⁺.

Step-3:N-Hydroxy-2′-oxo-1′-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide

Into an 8-mL vial was placed a solution of2′-oxo-1′-(3-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylicacid (30 mg, 0.08 mmol, 1 equiv) in DMA (3 mL). This was followed by theaddition of isopropyl chloroformate (95 mg, 0.78 mmol, 10 equiv)dropwise with stirring at 0° C. To this was added NMM (78 mg, 0.77 mmol,10 equiv) dropwise with stirring at 0° C. The mixture was stirring for 5min. To the mixture was added a solution of NH₂OH.HCl (54 mg, 0.78 mmol,10 equiv) in DMA (1 mL) dropwise with stirring at 0° C. The resultingsolution was stirred for 24 h at room temperature. The crude product waspurified by Prep-HPLC with the following conditions: Column: XBridgePrep C18 OBD, 19×150 mm, 5 μm; Mobile Phase A: water/0.1% formic acid,Mobile Phase B: CH₃CN; Gradient: 20% B to 41% B in 8 min, hold at 41% Bfor 4 min; Detector: UV 254 nm, 220 nm. The collected fractions werelyophilized to give 12.6 mg (40% yield) of the title compound as anorange solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 10.84 (br s, 1H), 9.04(br s, 1H), 8.27 (s, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.67-7.63 (m, 1H),7.51 (d, J=7.6 Hz, 1H), 7.24-7.09 (m, 3H), 3.95-3.84 (m, 2H), 2.97-2.74(m, 4H), 2.09-2.03 (m, 1H), 1.95-1.79 (m, 3H). MS: (ES, m/z): 405[M+H]⁺.

Example 24-2—Preparation ofN-hydroxy-2′-oxo-1′-(2-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide(II-73)

Step-1: Methyl2′-oxo-1′-(2-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate

Into a 20-mL microwave tube purged and maintained with an inertatmosphere of nitrogen, was placed a solution of methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(100 mg, 0.39 mmol, 1 equiv) in toluene (5 mL),1-iodo-2-(trifluoromethyl)benzene (210 mg, 0.77 mmol, 2 equiv), CuI (7mg, 0.04 mmol, 0.1 equiv), methyl[2-(methylamino)ethyl]amine (7 mg, 0.08mmol, 0.2 equiv) and K₃PO₄ (250 mg, 1.18 mmol, 3 equiv). The resultingsolution was stirred overnight at 110° C. The reaction mixture wascooled to room temperature and then poured into 20 mL of water. Theresulting solution was extracted with 3×20 mL of EtOAc. The combinedorganic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The residue was purified by preparative TLCwith EtOAc/petroleum ether (1:3). The collected fractions wereconcentrated under vacuum to give 50 mg (32% yield) of the titlecompound as yellow oil. MS: (ES, m/z): 404 [M+H]⁺.

Step-2:N-Hydroxy-2′-oxo-1′-(2-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxamide

Into a 10-mL round-bottom flask was placed a solution of methyl2′-oxo-1′-(2-(trifluoromethyl)phenyl)-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-8-carboxylate(50 mg, 0.12 mmol, 1 equiv), THF/MeOH (4:1, 3 mL), NH₂OH (50% in H₂O,491 mg, 7.2 mmol, 60 equiv), and aq. 1N NaOH (0.24 mL, 0.24 mmol, 2equiv). The resulting solution was stirred for 2 h at room temperature.The solids were filtered out. The crude product was purified byPrep-HPLC with the following conditions: Column: XBridge Prep C18 OBD,19×150 mm, 5 μm; Mobile Phase A: water/10 mM NH₄HCO₃, Mobile Phase B:CH₃CN; Gradient: 30% B to 40% B in 7 min; Detector: UV 254 nm, 220 nm.The collected fractions were lyophilized to give 11.5 mg (23% yield) ofthe title compound as a white solid. ¹H NMR (300 MHz, DMSO-d6) δ (ppm):10.80 (br s, 1H), 9.05 (br s, 1H), 7.85-7.77 (m, 2H), 7.65-7.52 (m, 2H),7.23-7.09 (m, 3H), 3.69-3.59 (m, 2H), 3.01-2.77 (m, 4H), 2.17-2.07 (m,1H), 1.91-1.81 (m, 3H). MS: (ES, m/z): 405 [M+H]⁺.

TABLE 8-2 Found (ES, m/z) Ex. Structure ¹H-NMR δ (ppm) [M + H]⁺ II-90

(300 MHz, DMSO-d6): 10.81 (br s, 1H), 9.04 (br s, 1H), 7.85-7.78 (m,2H), 7.65-7.54 (m, 2H), 7.24-7.09 (m, 3H), 3.70-3.65 (m, 2H), 2.98-2.83(m, 4H), 2.13-2.05 (m, 1H), 1.93-1.81 (m, 3H) 405The following compound was prepared according to the method of Example24-2, with the following modification: In Step 1, methyl2′-oxo-3,4-dihydro-1H-spiro[naphthalene-2,3′-pyrrolidine]-5-carboxylatewas used.

Example 25-2—Preparation of methylspiro[chromane-2,4′-piperidine]-8-carboxylate (Intermediate)

Step-1: 1′-(tert-Butyl) 8-methyl4-oxospiro[chromane-2,4′-piperidine]-1′,8-dicarboxylate

N-tert-Butoxycarbonyl-4-piperidone (1.0 g, 5.0 mmol, 1 equiv) was addedto a stirred solution of methyl 3-acetyl-2-hydroxybenzoate (0.97 g, 5.0mmol, 1 equiv) and pyrrolidine (418 μL, 5.0 mmol, 1 equiv) in MeOH (10mL) at room temperature. The reaction mixture was stirred at roomtemperature for 24 h. The reaction mixture was then concentrated andpartitioned between EtOAc (75 mL) and 2N HCl (75 mL). The layers wereseparated and the aqueous layer was extracted with EtOAc (2×150 mL). Thecombined organic layers were washed with water (50 mL) and brine (50mL), dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The crude product was purified by flash column chromatographyon silica gel (30% EtOAc/hexanes) to afford 1.71 g (91% yield) of thetitle compound as a pale yellow solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm):8.08-8.04 (m, 2H), 7.05 (t, J=7.4 Hz, 1H), 3.96 (br s, 2H), 3.91 (s,3H), 3.28 (br s, 2H), 2.75 (s, 2H), 2.05 (app d, J=12.5 Hz, 2H), 1.59(dt, J=13.2, 4.9 Hz, 2H), 1.45 (s, 9H). MS: (ES, m/z): 276 [M+H-Boc]⁺.

Step-2: 1′-(tert-Butyl) 8-methyl4-hydroxyspiro[chromane-2,4′-piperidine]-1′,8-dicarboxylate

NaBH₄ (190 mg, 5.01 mmol, 1.1 equiv) was added to a stirred solution of1′-(tert-butyl)-8-methyl-4-oxospiro[chromane-2,4′-piperidine]-1′,8-dicarboxylate(1.71 g, 4.55 mmol, 1 equiv) in MeOH (15 mL), cooled to 0° C. Thereaction warmed to room temperature over 15 min, then was quenched with60 mL of aq. sat. NH₄Cl solution. The mixture was extracted with EtOAc(4×50 mL) and dried over Na₂SO₄. The Na₂SO₄ was filtered off and thesolvent was removed in vacuo to afford the title compound as anoff-white solid, which was carried forward without further purification.

Step-3: Methyl spiro[chromane-2,4′-piperidine]-8-carboxylate

1′-(tert-Butyl)-8-methyl-4-hydroxyspiro[chromane-2,4′-piperidine]-1′,8-dicarboxylatewas dissolved in 3 mL of triethylsilane. Then 2 mL of TFA was added. Themixture was stirred for 48 h at room temperature. The reaction mixturewas then diluted with EtOAc (100 mL) and the excess acid was quenchedwith aq. 10% K₂CO₃ (30 mL). Then layers were separated and the organicphase was dried over Na₂SO₄. After filtration, the organic phase wasreduced to 10 mL and 4N HCl in dioxane was added. A white precipitateformed. Hexanes (50 mL) was added and the mixture was triturated andfiltered to afford 1.48 g (85% yield over 2 steps) of the title compoundas the TFA salt as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm):8.87 (br s, 1H), 8.55 (br s, 1H), 7.54 (d, J=9.0 Hz, 1H), 7.33 (d, J=7.4Hz, 1H), 6.93 (t, J=7.8 Hz, 1H), 3.82 (s, 3H), 3.26-3.14 (m, 4H), 2.82(t, J=6.6 Hz, 2H), 1.92-1.75 (m, 6H). ¹³C NMR (100 MHz, DMSO-d6) δ(ppm): 166.3, 151.6, 133.8, 129.1, 122.8, 119.8, 119.4, 71.8, 51.9,30.6, 30.1, 20.6. MS: (ES, m/z): 262 [M+H-TFA]⁺.

Example 26-2—Preparation of methylspiro[chromane-2,4′-piperidine]-5-carboxylate (Intermediate)

Step-1: tert-Butyl5-bromo-4-oxospiro[chromane-2,4′-piperidine]-1′-carboxylate

N-tert-butoxycarbonyl-4-piperidone (0.996 g, 5 mmol) was added to astirred solution of 1-(2-bromo-6-hydroxyphenyl)ethanone (1.08 g, 5 mmol)in 5 mL of MeOH, followed by pyrrolidine (0.82 mL, 10 mmol). Thereaction mixture was sealed and followed at room temperature for 48 h.The mixture was then diluted with 75 mL of EtOAc and washed with 1N HCl(2×50 mL) and brine (1×50 mL). The organic phase was dried over Na₂SO₄.After filtration, the organic solvent was removed in vacuo. The viscousred residue was purified by flash column chromatography on silica gel(40% EtOAc/hexanes) to afford 1.19 g (60% yield) of the title compoundas an off-white solid. ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 7.41 (t, J=8.2Hz, 1H), 7.28 (dd, J=8.2, 1.0 Hz, 1H), 7.10 (dd, J=8.6, 1.0 Hz, 1H),3.69 (app d, 2H), 3.12 (br s, 2H), 2.88 (s, 2H), 1.85-1.82 (m, 2H),1.66-1.58 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ (ppm): 189.8, 160.2,153.8, 153.9, 127.6, 119.8, 118.4, 118.3, 78.8, 77.8, 47.3, 32.9, 28.1.MS: (ES, m/z): 296 [M+H-Boc]⁺.

Step-2: 1′-(tert-Butyl) 5-methyl4-oxospiro[chromane-2,4′-piperidine]-1′,5-dicarboxylate

tert-Butyl-5-bromo-4-oxospiro[chromane-2,4′-piperidine]-1′-carboxylate(1.31 g, 3.3 mmol) was added to a stirred solution of palladium (II)chloride (47 mg, 0.26 mmol) and 1,3-bis(diphenylphosphanyl)propane (110mg, 0.26 mmol) in anhydrous MeOH (10 mL) and anhydrous DMF (1.5 mL) in apressure vessel. CO (g) was vigorously bubbled through the reactionmixture while Et₃N (1.5 mL) was added. The vessel was sealed under CO(g) and heated at 75° C. for 24 h. The reaction mixture was then cooledto room temperature and diluted with EtOAc (100 mL) and washed with 1MHCl (2×50 mL) and brine (1×50 mL). The organic phase was dried overNa₂SO₄. After filtration, the organic solvent was removed in vacuo andthe residue was purified by flash column chromatography on silica gel(40% EtOAc/hexanes) to afford 400 mg (32% yield) of the title compoundas an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.50 (dd, J=8.2,0.8 Hz, 1H), 7.07 (dd, J=8.2, 0.8 Hz, 1H), 6.97 (dd, J=7.4, 1.2 Hz, 1H),3.93 (s, 3H), 3.87 (br s, 2H), 3.21 (app t, J=11.7 Hz, 2H), 2.75 (s,2H), 2.02 (app d, J=13.3 Hz, 2H), 1.62 (dt, J=12.9, 5.1 Hz, 2H), 1.46(s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 190.1, 169.8, 158.9, 154.6,135.7, 133.9, 120.0, 117.7, 79.8, 78.3, 52.8, 47.9, 33.9, 28.4. MS: (ES,m/z): 398 [M+Na]⁺.

Step-3: 1′-(tert-Butyl) 5-methyl4-hydroxyspiro[chromane-2,4′-piperidine]-1′,5-dicarboxylate

NaBH₄ (161 mg, 4.28 mmol) was added to a stirred solution of1′-(tert-butyl)-5-methyl-4-oxospiro[chromane-2,4′-piperidine]-1′,5-dicarboxylate(0.400 g, 1.07 mmol) in 10 mL of MeOH, cooled to 0° C. The reactionwarmed to room temperature over 15 min, then was quenched with 60 mL ofaq. sat. NH₄Cl solution. The mixture was extracted with EtOAc (4×50 mL)and dried over Na₂SO₄. The Na₂SO₄ was filtered off and the solvent wasremoved in vacuo to afford the title compound as an off-white solid,which was carried forward without further purification.

Step-4: Methyl spiro[chromane-2,4′-piperidine]-5-carboxylate

1′-(tert-Butyl)-5-methyl-4-hydroxyspiro[chromane-2,4′-piperidine]-1′,5-dicarboxylatewas dissolved in 3 mL of triethylsilane. Then 2 mL of TFA was added. Themixture stirred for 48 h at room temperature. The reaction mixture wasthen diluted with EtOAc (100 mL) and the excess acid was quenched withaq. 10% K₂CO₃ (30 mL). The layers were separated and the organic phasewas dried over Na₂SO₄. After filtration, the organic phase wasconcentrated to 10 mL and 4N HCl in dioxane was added. A whiteprecipitate formed. 50 mL of hexanes was added and the mixture wastriturated and filtered to afford 236 mg (75% yield over 2 steps) of thetitle compound as the HCl salt as a white solid. ¹H NMR (400 MHz, CDCl₃)δ (ppm): 10.0 (br s, 1H), 8.63 (br s, 1H), 7.54 (dd, J=7.8, 1.1 Hz, 1H),7.19 (t, J=8.2 Hz, 1H), 7.04 (dd, J=7.8, 1.0 Hz, 1H), 3.88 (s, 3H), 3.23(br s, 4H), 3.13 (t, J=7.0 Hz, 2H), 1.98 (br s, 4H), 1.88 (t, J=7.0 Hz,2H). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 167.5, 152.9, 130.2, 127.1,123.4, 122.9, 121.7, 70.5, 51.9, 39.3, 31.6, 31.2, 20.1. MS: (ES, m/z):262 [M+H]⁺.

Example 27-2—Preparation ofN8-hydroxy-N1′,N1′-dimethylspiro[chromane-2,4′-piperidine]-1′,8-dicarboxamide(II-74)

Step-1: Methyl1′-(dimethylcarbamoyl)spiro[chromane-2,4′-piperidine]-8-carboxylate

A 2-mL vial was charged with a solution of methylspiro[chromane-2,4′-piperidine]-8-carboxylate TFA (0.2M in DCE/DIPEA(10:1), 200 μL, 0.040 mmol, 1 equiv) followed by dimethylcarbamylchloride (0.2M in DCE, 200 μL, 0.040 mmol, 1 equiv). The vial was sealedand shaken at room temperature for 18 h, then the solvent was removedunder a stream of N₂ (g). The residue was diluted with brine (500 μL)and extracted with EtOAc (2×500 μL). The combined organic layers weredried under a stream of N₂ (g) revealing a pale yellow residue, whichwas used without further purification. ¹H NMR (400 MHz, CDCl₃) δ (ppm):7.65 (dd, J=7.8, 1.6 Hz, 1H), 7.20 (d, J=7.4 Hz, 1H), 6.86 (t, J=7.4 Hz,1H), 3.89 (s, 3H), 3.57-3.52 (m, 2H), 3.67 (dt, J=12.5, 2.0 Hz, 2H),2.84-2.80 (m, 8H), 1.84-1.79 (m, 4H), 1.67-1.59 (m, 2H). MS: (ES, m/z):333 [M+H]⁺.

Step-2:N8-Hydroxy-N1′,N1′-dimethylspiro[chromane-2,4′-piperidine]-1′,8-dicarboxamide

The residue from Step 1 was dissolved in THF/MeOH (3:1, 200 μL). Thevial was sealed and shaken at 50° C. for 15 min to dissolve the residue,then cooled to room temperature. NH₂OH (50% v/v solution in water, 150μL) was added, followed by aq. 1N NaOH (100 μL). The mixture was sealedand shaken at room temperature for 18 h. The reaction mixture wasconcentrated under a stream of N₂ (g) at room temperature, thendissolved in 500 μL of DMSO and purified by Prep-HPLC using thefollowing conditions: Column: Waters Sunfire C18, 19×50 mm, 5 μm; MobilePhase A: water/0.1% formic acid, Mobile Phase B: CH₃CN/0.1% formic acid;Gradient: 15% B up to 100% B in 6 min; Flow Rate: 23 mL/min; Detector:UV 254 nm, 220 nm. The product-containing fractions were combined andconcentrated to afford 2.5 mg (19% over 2 steps) of the title compound.¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.08 (s, 1H), 8.01 (dd, J=7.8, 1.6 Hz,1H), 7.23 (d, J=7.4 Hz, 1H), 7.01 (t, J=7.8 Hz, 1H), 3.55 (dt, J=13.7,3.9 Hz, 1H), 3.22 (ddd, J=13.9, 11.3, 2.9 Hz, 1H), 2.88-2.83 (m, 8H),1.94-1.88 (m, 4H), 1.81-1.73 (m, 2H). MS: (ES, m/z): 334 [M+H]⁺.

TABLE 9-2 Found (ES, m/z) Ex. Structure [M + H]⁺ II-75

385 II-76

409 II-77

389 II-78

475 II-79

381The following compounds were prepared according to the parallelsynthesis method of Example 27-2.

TABLE 10-2 Found (ES, m/z) Ex. Structure [M + H]⁺ II-91

305The following compound was prepared according to the parallel synthesismethod of Example 27-2, with the following modification: In Step 1,methyl spiro[chromane-2,4′-piperidine]-5-carboxylate HCl was used.

Example 28-2—Preparation of1′-(4-fluorobenzyl)-N-hydroxyspiro[chromane-2,4′-piperidine]-8-carboxamide(II-80)

Step-1: Methyl1′-(4-fluorobenzyl)spiro[chromane-2,4′-piperidine]-8-carboxylate

A 2-mL vial was charged with a solution of methylspiro[chromane-2,4′-piperidine]-8-carboxylate TFA (0.2M in DCE/DIPEA(10:1), 200 μL, 0.040 mmol, 1 equiv), followed by a solution of4-fluorobenzaldehyde (0.2M in DCE, 200 μL, 0.040 mmol, 1 equiv). ThenNaBH(OAc)₃ (42 mg, 0.080 mmol, 2 equiv) was added in one portion. Thevial was sealed and shaken at room temperature for 18 h, then thesolvent was removed under a stream of N₂ (g). The residue was dilutedwith brine (500 μL) and extracted with EtOAc (2×500 μL). The combinedorganic layers were dried under a stream of N₂ (g) revealing a paleyellow residue, which was used without further purification. ¹H NMR (400MHz, CDCl₃) δ (ppm): 7.64 (dd, J=7.8, 2.0 Hz, 1H), 7.41 (dd, J=8.4, 5.3Hz, 2H), 7.20 (d, J=7.8 Hz, 1H), 7.05 (t, J=8.6 Hz, 2H), 6.87 (t, J=7.6Hz, 1H), 3.88 (s, 3H), 3.84 (br s, 2H), 2.97-2.86 (m, 4H), 2.82 (t,J=8.6 Hz, 2H), 1.96-1.85 (m, 6H). MS: (ES, m/z): 370 [M+H]⁺.

Step-2:1′-(4-Fluorobenzyl)-N-hydroxyspiro[chromane-2,4′-piperidine]-8-carboxamide

The residue from Step 1 was dissolved in THF/MeOH (3:1, 200 μL). Thevial was sealed and shaken at 50° C. for 15 min to dissolve the residue,then cooled to room temperature. NH₂OH (50% v/v solution in water, 150μL) was added, followed by aq. 1N NaOH (100 μL). The mixture was sealedand shaken at room temperature for 18 h. The reaction mixture wasconcentrated under a stream of N₂ (g) at room temperature, thendissolved in 500 μL of DMSO and purified by Prep-HPLC using thefollowing conditions: Column: Waters Sunfire C18, 19×50 mm, 5 μm; MobilePhase A: water/0.1% formic acid, Mobile Phase B: CH₃CN/0.1% formic acid;Gradient: 15% B up to 100% B in 6 min; Flow Rate: 23 mL/min; Detector:UV 254 nm, 220 nm. The product-containing fractions were combined andconcentrated to afford the title compound. MS: (ES, m/z): 371 [M+H]⁺.

TABLE 11-2 Found (ES, m/z) Ex. Structure [M + H]⁺ II-81

345The following compound was prepared according to the parallel synthesismethod of Example 28-2.

Example 29-2—Preparation ofN-hydroxy-1′-(5-(trifluoromethyl)pyridin-2-yl)spiro[chromane-2,4′-piperidine]-8-carboxamide(II-82)

Step-1: Methyl1′-(5-(trifluoromethyl)pyridin-2-yl)spiro[chromane-2,4′-piperidine]-8-carboxylate

A 2-mL vial was charged methylspiro[chromane-2,4′-piperidine]-8-carboxylate TFA (0.2M in dioxane, 200μL, 40 μmol, 1 equiv) and Cs₂CO₃ (39 mg, 120 μmol, 3 equiv). Then asolution of 2-chloro-5-(trifluoromethyl)pyridine (0.2M in dioxane, 400μL, 80 μmol, 2 equiv) was added. The vial was sealed and brought into aglovebox. Then a degassed solution of the copper(I) bromide andN,N-dimethylethane-1,2-diamine ligand (0.02M in DMA, 150 μL, 3.0 mol)was added. The vial was sealed and heated at 105° C. for 18 h. Thesolvent was removed under a stream of N₂ (g). The residue was dilutedwith brine (500 μL) and extracted with EtOAc (2×500 μL). The combinedorganic layers were dried under a stream of N₂ (g). The residue wascarried forward without further purification. ¹H NMR (400 MHz, CDCl₃) δ(ppm): 8.39 (s, 1H), 7.67 (dd, J=7.8, 2.0 Hz, 1H), 7.60 (dd, J=9.0, 2.3Hz, 1H), 7.22 (d, J=7.4 Hz, 1H), 6.89 (t, J=7.8 Hz, 1H), 6.68 (d, J=9.0Hz, 1H), (dt, J=13.4, 2.3 Hz, 2H), 3.87 (s, 3H), 3.59-3.51 (m, 2H), 2.86(t, J=6.8 Hz, 2H), 1.93 (dd, J=14.3, 2.2 Hz, 2H), 1.85 (t, J=7.0 Hz,2H), 1.67-1.59 (m, 2H). MS: (ES, m/z): 407 [M+H]⁺.

Step-2:N-Hydroxy-1′-(5-(trifluoromethyl)pyridin-2-yl)spiro[chromane-2,4′-piperidine]-8-carboxamide

The residue from Step 1 was taken up in THF/MeOH (3:1, 200 μL). The vialwas sealed and shaken at 50° C. for 15 min to dissolve the residue, thencooled to room temperature. NH₂OH (50% v/v solution in water, 150 μL)was added, followed by aq. 1N NaOH (100 μL). The mixture was sealed andthen shaken at room temperature for 18 h. The reaction mixture wasconcentrated under a stream of N₂ (g) at room temperature, thendissolved in 500 μL of DMSO and purified by Prep-HPLC using thefollowing conditions: Column: Waters Sunfire C18, 19×50 mm, 5 μm; MobilePhase A: water/0.1% formic acid, Mobile Phase B: CH₃CN/0.1% formic acid;Gradient: 15% B up to 100% B in 6 min; Flow Rate: 23 mL/min; Detector:UV 254 nm, 220 nm. The product-containing fractions were combined andconcentrated to afford 4.0 mg (25% yield) of the title compound. ¹H NMR(400 MHz, DMSO-d6) δ (ppm): 10.52 (s, 1H), 9.07 (s, 1H), 8.41 (s, 7.78(dd, J=9.2, 2.5 Hz, 1H), 7.27 (dd, J=7.6, 1.4 Hz, 1H), 7.20 (d, J=7.8Hz, 1H), 7.01 (d, J=9.4 Hz, 1H), 6.89 (t, J=7.4 Hz, 1H), 4.23 (d, J=13.3Hz, 2H), 3.39 (t, J=11.3 Hz, 2H), 2.79 (t, J=6.6 Hz, 2H), 1.84-1.80 (m,4H), 1.65-1.57 (m, 2H). MS: (ES, m/z): 408 [M+H]⁺.

TABLE 12-2 Found (ES, m/z) Ex. Structure [M + H]⁺ II-83

371The following compound was prepared according to the parallel synthesismethod of Example 29-2.

TABLE 13-2 Found (ES, m/z) Ex. Structure [M + H]⁺ II-92

339 II-93

357The following compounds were prepared according to the parallelsynthesis method of Example 29-2, with the following modification: InStep 1, methyl spiro[chromane-2,4′-piperidine]-5-carboxylate HCl wasused.

Example 30-2—In Vitro Histone Deacetylase Assay

The enzymatic HDAC11 assay was performed using electrophoretic mobilityshift assay. Full length human recombinant HDAC11 protein was expressedin baculoviral system and purified by affinity chromatography. Theenzymatic reactions were assembled in 384 well plates in a total volumeof 25 μL in a reaction buffer composing: 100 mM HEPES, pH 7.5, 25 mMKCl, 0.1% bovine serum albumin, 0.01% Triton X-100, 1% DMSO (fromcompounds) 2 M of the fluorescently labeled peptide substrate andenzyme. The enzyme was added at a final concentration of 10 nM. Thepeptide substrate FAM-RHKK(tri-fluor-Ac)-NH₂ was used. The compoundswere tested at 12 concentrations spaced by 3× dilution intervals.Negative control samples (0%−inhibition in the absence of inhibitor) andpositive control samples (100%−inhibition) were assembled in replicatesof four in each assay plate. The reactions were incubated at 25° C. andquenched by the addition of 45 μL of termination buffer (100 mM HEPES,pH 7.5, 0.01% Triton X-100, 0.05% SDS).

The terminated assay plates were analyzed on LabChip® 3000 microfluidicelectrophoresis instrument (Perkin Elmer/Caliper Life Sciences). Thefluorescence intensity of the electrophoretically separatedde-acetylated product and substrate peptide was measured. Activity ineach sample was determined as the product to sum ratio (PSR): P/(S+P),where P is the peak height of the product peptide and S is the peakheight of the substrate peptide. Percent inhibition (P_(inh)) isdetermined using the following equation:

P _(inh)=(PSR0%−PSR_(inh))/(PSR0%−PSR100%)*100,

where PSR_(inh) is the product sum ratio in the presence of inhibitor,PSR0% is the average product sum ration in the absence of inhibitor andPSR100% is the average product sum ratio in 100%−inhibition controlsamples. The IC₅₀ values of inhibitors were determined by fitting thepercent inhibition curves with 4 parameter dose-response model usingXLfit 4 software.

As set forth in Table 14-2 below, “+++” indicates an IC₅₀ below 0.5 μM;“++” indicates an IC₅₀ between 0.5 μM and 1 μM; and “+” indicates anIC₅₀ above 1 μM.

TABLE 14-2 IC₅₀ Ranges for Compounds of the Disclosure Compound HDAC11No. IC₅₀ Range II-1 +++ II-2 + II-3 +++ II-4 +++ II-5 +++ II-6 +++ II-7++ II-8 + II-9 + II-10 + II-11 + II-12 ++ II-13 + II-14 + II-15 + II-16+++ II-17 ++ II-18 +++ II-19 + II-20 + II-21 ++ II-22 +++ II-23 +II-24 + II-25 + II-26 + II-27 +++ II-28 + II-29 ++ II-30 +++ II-31 +++II-32 ++ II-33 +++ II-34 + II-35 + II-36 + II-37 +++ I-38 +++ II-39 ++II-40 + II-41 +++ II-42 +++ II-43 +++ II-44 + II-45 ++ II-46 + II-47 +II-48 + II-49 + II-50 + II-51 + II-52 + II-53 + II-54 + II-55 + II-56 ++II-57 + II-58 + II-59 + II-60 + II-61 + II-62 + II-63 + II-64 + II-65 +II-66 + II-67 +++ II-68 +++ II-69 + II-70 ++ II-71 ++ II-72 + II-73 +II-74 + II-75 + II-76 + II-77 + II-78 +++ II-79 + II-80 + II-81 +II-82 + II-83 + II-84 +++ II-85 +++ II-86 +++ II-87 + II-88 +++ II-89 ++II-90 + II-91 + II-92 + II-93 +

1.-49. (canceled)
 50. A method of treating cancer in a patient in needthereof, comprising administering to a patient an effective amount of aHDAC11 inhibitor.
 51. The method of claim 50, wherein one or more cancercells in the patient exhibit stem cell-like properties.
 52. The methodof claim 50, wherein the cancer is a hematological cancer.
 53. A methodof treating a patient having a myeloproliferative disorder, comprisingadministering to the patient a HDAC11 inhibitor and a second therapeuticagent.
 54. The method of claim 53, wherein the second therapeutic agentis a JAK2 inhibitor.
 55. The method of claim 53, wherein themyeloproliferative disorder is resistant to a JAK2 inhibitor.
 56. Themethod of claim 53, wherein the second therapeutic agent is a hedgehogpathway inhibitor.
 57. The method of claim 50, wherein the patient hasreceived a first line therapy, and any cancer cells surviving from thefirst line therapy are reduced or eliminated after treatment with theHDAC11 inhibitor.
 58. A compound of Formula I′ or a pharmaceuticallyacceptable salt thereof,

wherein: Q is —H, —OC(O)NR⁶(C₁-C₆)alkylaryl, or —OC(O)O(C₁-C₆)alkylaryl;Z is —CH₂—, O, S or NR⁶; X₁, X₂, X₃, and X₄ are each independently, ateach occurrence, N or CR¹; Y¹, Y², Y³, and Y⁴ are each independently Nor CR¹; L is NR⁶, O, or —(CR¹R²)_(p)—; R¹ and R² are independently, ateach occurrence, —H, —R³, —R⁴, —C₁-C₆alkyl, —C₂-C₆alkenyl,—C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,heteroaryl containing 1-5 heteroatoms selected from the group consistingof N, S, P and O, —OH, —OR³, halogen, —NO₂, —CN, —NHC₁-C₆alkyl,—N(C₁-C₆alkyl)₂, —S(O)₂NH₂, —S(O)₂N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)₂R⁵,—S(O)₂(C₁-C₆alkyl), —(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl,—C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, or —(CHR⁵)_(p)NR³R⁴,wherein each alkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkyl,heterocyclyl, aryl, or heteroaryl is optionally substituted with one ormore —OH, halogen, —NO₂, oxo, —CN, —R³, —R⁵, —SR³, —OR³, —NHR³, —NR³R⁴,—S(O)₂N(R³)₂—, —S(O)₂R⁵, —C(O)R⁵, —C(O)OR⁵, —NR³S(O)₂R⁵, —S(O)R⁵,—S(O)NR³R⁴, —NR³S(O)R⁵, heterocycle, aryl, or heteroaryl; or R¹ and R²can combine with the carbon atom to which they are both attached to forma spirocycle, spiroheterocycle, or spirocycloalkenyl, each optionallysubstituted with one or more independent occurrences of R³ and R⁴; ortwo occurrences of R¹, when on adjacent atoms, can combine to form acycloalkyl, a heterocycle, aryl, heteroaryl containing 1-5 heteroatomsselected from the group consisting of N, S, P and O, or a cycloalkenyl,each optionally substituted with one or more independent occurrences ofR³ and R⁴; R³ and R⁴ are independently, at each occurrence, —H,—C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,—C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5heteroatoms selected from N, S, P, and O, —S(O)₂N(C₁-C₆alkyl)₂,—S(O)₂(C₁-C₆alkyl), —(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl,—C(O)OC₁-C₆alkyl, oxo, or —(CHR⁵)_(p)N(C₁-C₆alkyl)₂, wherein each alkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, andheteroaryl is optionally substituted with one or more substituentsselected from —OH, halogen, —NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl,—NH(C₁-C₆)alkyl, —N(C₁-C₆alkly)₂, —S(O)₂N(C₁-C₆alkyl)₂,—S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,—N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵, —S(O)N(C₁-C₆alkyl)₂,—N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, or heteroaryl; R⁵ isindependently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆alkenyl,—C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,heteroaryl containing 1-5 heteroatoms selected from N, S, P and O, —OH,halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,—S(O)₂NH(C₁-C₆alkyl), —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂C₁-C₆alkyl,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)SO₂C₁-C₆alkyl,—S(O)(C₁-C₆alkyl), —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)(C₁-C₆alkyl)or —(CH₂)_(p)N(C₁-C₆alkyl)₂; R⁶ is independently, at each occurrence,—H, —C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,—C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5heteroatoms selected from the group consisting of N, S, P and O,—S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂(C₁-C₆alkyl), —(C₁-C₆alkyl)S(O)₂R⁵,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, or —(CHR⁵)_(p)NR³R⁴ wherein eachalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,and heteroaryl is optionally substituted with one or more substituentsselected from —OH, halogen, —NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl,—NH(C₁-C₆)alkyl, —N(C₁-C₆alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂,—S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,—N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵, —S(O)N(C₁-C₆alkyl)₂,—N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, or heteroaryl; and p is 0, 1,2, 3, 4, 5, or
 6. 59. A compound of Formula II or a pharmaceuticallyacceptable salt thereof.

and pharmaceutically acceptable salts thereof, wherein: X¹, X², X³, X⁴,X⁵, and X⁶ are each independently, at each occurrence, —CR¹R²—, —NR³—,—O—, —C(O)—, —S(O)₂—, —S(O)—, or —S—; Y¹, Y², and Y³ are eachindependently N or CR¹; L is a bond, —(CR¹R²)_(p)—, —C(O)NR³—, —S(O)₂—,—S(O)₂NR³—, —S(O)—, —S(O)NR³—, —C(O)(CR¹R²)_(p)O—, or —C(O)(CR¹R²)_(p)—;R is independently —H, —C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl,—C₂-C₆alkynyl, —C₃-C₈cycloalkyl, —C₅-C₁₂spirocycle, heterocyclyl,spiroheterocyclyl, aryl, or heteroaryl containing 1-5 heteroatomsselected from the group consisting of N, S, P, or O, wherein each alkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkyl, spirocycle, heterocyclyl,spiroheterocyclyl, aryl, or heteroaryl is optionally substituted withone or more —OH, halogen, oxo, —NO₂, —CN, —R¹, —R², —SR³, —OR³, —NHR³,—NR³R⁴, —S(O)₂NR³R⁴, —S(O)₂R¹, —C(O)R¹, —C(O)OR¹, —NR³S(O)₂R¹, —S(O)R¹,—S(O)NR³R⁴, —NR³S(O)R¹, heterocycle, aryl, or heteroaryl; R¹ and R² areindependently, at each occurrence, —H, —R³, —R⁴, —C₁-C₆alkyl,—C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl,heterocyclyl, aryl, heteroaryl containing 1-5 heteroatoms selected fromthe group consisting of N, S, P and O, —OH, halogen, —NO₂, —CN,—NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂, —S(O)₂N(C₁-C₆alkyl)₂,—N(C₁-C₆alkyl)S(O)₂R⁵, —S(O)₂(C₁-C₆alkyl), —(C₁-C₆alkyl)S(O)₂R⁵,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, or—(CHR⁵)_(p)NR³R⁴, wherein each alkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substitutedwith one or more —OH, halogen, —NO₂, oxo, —CN, —R⁵, —OR³, —NHR³, —NR³R⁴,—S(O)₂N(R³)₂—, —S(O)₂R⁵, —C(O)R⁵, —C(O)OR⁵, —NR³S(O)₂R⁵, —S(O)R⁵,—S(O)NR³R⁴, —NR³S(O)R⁵, heterocycle, aryl, or heteroaryl; or R¹ and R²can combine with the carbon atom to which they are both attached to forma spirocycle, spiroheterocycle, or spirocycloalkenyl; or R¹ and R², whenon adjacent atoms, can combine to form a cycloalkyl, a heterocycle,aryl, heteroaryl containing 1-5 heteroatoms selected from the groupconsisting of N, S, P and O, or a cycloalkenyl; or R¹ and R², when onnon-adjacent atoms, can combine to form an optionally bridgingcycloalkyl, an optionally bridging heterocycle, or an optionallybridging cycloalkenyl; R³ and R⁴ are independently, at each occurrence,—H, —C₁-C₆alkyl, —C₂-C₆alkenyl, —C₄-C₈cycloalkenyl, —C₂-C₆alkynyl,—C₃-C₈cycloalkyl, heterocyclyl, aryl, heteroaryl containing 1-5heteroatoms selected from N, S, P, and O, —S(O)₂N(C₁-C₆alkyl)₂,—S(O)₂(C₁-C₆alkyl), —(C₁-C₆alkyl)S(O)₂R⁵, —C(O)C₁-C₆alkyl,—C(O)OC₁-C₆alkyl, or —(CHR⁵)_(p)N(C₁-C₆alkyl)₂, wherein each alkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, andheteroaryl is optionally substituted with one or more substituentsselected from —OH, halogen, —NO₂, oxo, —CN, —R⁵, —O(C₁-C₆)alkyl,—NH(C₁-C₆)alkyl, —N(C₁-C₆alkly)₂, —S(O)₂N(C₁-C₆alkyl)₂,—S(O)₂NHC₁-C₆alkyl, —C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl,—N(C₁-C₆alkyl)S(O)₂C₁-C₆alkyl, —S(O)R⁵, —S(O)N(C₁-C₆alkyl)₂,—N(C₁-C₆alkyl)S(O)R⁵, heterocycle, aryl, or heteroaryl; R⁵ isindependently, at each occurrence, —H, —C₁-C₆alkyl, —C₂-C₆alkenyl,—C₄-C₈cycloalkenyl, —C₂-C₆alkynyl, —C₃-C₈cycloalkyl, heterocyclyl, aryl,heteroaryl containing 1-5 heteroatoms selected from N, S, P and O, —OH,halogen, —NO₂, —CN, —NHC₁-C₆alkyl, —N(C₁-C₆alkyl)₂,—S(O)₂NH(C₁-C₆alkyl), —S(O)₂N(C₁-C₆alkyl)₂, —S(O)₂C₁-C₆alkyl,—C(O)C₁-C₆alkyl, —C(O)OC₁-C₆alkyl, —N(C₁-C₆alkyl)SO₂C₁-C₆alkyl,—S(O)(C₁-C₆alkyl), —S(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆alkyl)S(O)(C₁-C₆alkyl)or —(CH₂)_(p)N(C₁-C₆alkyl)₂; p is 0, 1, 2, 3, 4, 5, or 6; n is 0, 1, 2,3, or 4; m is 0, 1, or 2; q is 1 or 2; r is 1 or 2; wherein the sumq+r≤3 and wherein the sum m+n≤4.